Cladribine analogues via O6-(benzotriazolyl) derivatives of guanine nucleosides

Article abstract of DOI:10.3390/molecules201018437

Cladribine, 2-chloro-2'-deoxyadenosine, is a highly efficacious, clinically used nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other lymphoid malignancies and has been a molecule of interest for well over half a century. In continuation of our interest in the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate, we have evaluated the use of O⁶-(benzotriazol-1-yl)-2'-deoxyguanosine as a potential precursor to cladribine and its analogues. These compounds, after appropriate deprotection, were assessed for their biological activities, and the data are presented herein. Against hairy cell leukemia (HCL), T-cell lymphoma (TCL) and chronic lymphocytic leukemia (CLL), cladribine was the most active against all. The bromo analogue of cladribine showed comparable activity to the ribose analogue of cladribine against HCL, but was more active against TCL and CLL. The bromo ribose analogue of cladribine showed activity, but was the least active among the C6-NH₂-containing compounds. Substitution with alkyl groups at the exocyclic amino group appears detrimental to activity, and only the C6 piperidinyl cladribine analogue demonstrated any activity. Against adenocarcinoma MDA-MB-231 cells, cladribine and its ribose analogue were most active.

Full text of DOI:10.3390/molecules201018437

Molecules 2015, 20, 18437-18463; doi:10.3390/molecules201018437
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
6
Cladribine Analogues via O -(Benzotriazolyl) Derivatives of
Guanine Nucleosides
1,†
1,†
2,†
Sakilam Satishkumar , Prasanna K. Vuram , Siva Subrahmanyam Relangi ,
2
3
3
Venkateshwarlu Gurram , Hong Zhou , Robert J. Kreitman ,
4
1
2
Michelle M. Martínez Montemayor , Lijia Yang , Muralidharan Kaliyaperumal ,
2
2
1,
Somesh Sharma , Narender Pottabathini and Mahesh K. Lakshman *
1
Department of Chemistry, The City College and The City University of New York,
160 Convent Avenue, New York, NY 10031, USA; E-Mails: ssakilam@ccny.cuny.edu (S.S.);
vkumar@ccny.cuny.edu (P.K.V.); lyang@sci.ccny.cuny.edu (L.Y.)
2
Discovery and Analytical Services, GVK Biosciences Pvt. Ltd., 28A IDA Nacharam,
Hyderabad 500076, India; E-Mails: siva.relangi@gvkbio.com (S.S.R.);
venkateshwarlu.gurram@gvkbio.com (V.G.); muralidharan.k@gvkbio.com (M.K.);
somesh.sharma@gvkbio.com (S.S.); narender.pottabathini@gvkbio.com (N.P.)
Clinical Immunotherapy Section, Laboratory of Molecular Biology, National Cancer Institute,
National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA;
E-Mails: hong.zhou@nih.gov (H.Z.); kreitmar@mail.nih.gov (R.J.K.)
3
4
Department of Biochemistry, Universidad Central del Caribe-School of Medicine, P. O. Box 60327,
Bayamón, PR 00960, USA; E-Mail: mmmtz92@gmail.com
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: mlakshman@ccny.cuny.edu;
Tel.: +1-212-650-7835; Fax: +1-212-650-6107.
Academic Editor: Fumi Nagatsugi
Received: 17 August 2015 / Accepted: 22 September 2015 / Published: 9 October 2015
Abstract: Cladribine, 2-chloro-2′-deoxyadenosine, is a highly efficacious, clinically used
nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other
lymphoid malignancies and has been a molecule of interest for well over half a century.
In continuation of our interest in the amide bond-activation in purine nucleosides via the use
of (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate, we have
6
evaluated the use of O -(benzotriazol-1-yl)-2′-deoxyguanosine as a potential precursor to

Molecules 2015, 20
18438
cladribine and its analogues. These compounds, after appropriate deprotection, were assessed
for their biological activities, and the data are presented herein. Against hairy cell leukemia
HCL), T-cell lymphoma (TCL) and chronic lymphocytic leukemia (CLL), cladribine was the
(
most active against all. The bromo analogue of cladribine showed comparable activity to the
ribose analogue of cladribine against HCL, but was more active against TCL and CLL. The
bromo ribose analogue of cladribine showed activity, but was the least active among the
C6-NH -containing compounds. Substitution with alkyl groups at the exocyclic amino group
2
appears detrimental to activity, and only the C6 piperidinyl cladribine analogue demonstrated
any activity. Against adenocarcinoma MDA-MB-231 cells, cladribine and its ribose analogue
were most active.
Keywords: cladribine; nucleoside; guanosine; benzotriazole; (benzotriazol-1yl-oxy)-
tris(dimethylamino)phosphonium hexafluorophosphate; BOP
1
. Introduction
Cladribine, 2-chloro-2′-deoxyadenosine, has been a molecule of interest for well over five decades.
The history of this compound dates back to 1960, when it was used in the synthesis of 2′-deoxyguanosine
and 2′-deoxyinosine [1]. A decade later, cladribine was shown to be a poor substrate for adenosine
deaminase that underwent phosphorylation by deoxycytidine kinase, finally resulting in the triphosphate,
and inhibiting DNA synthesis rather than RNA synthesis [2,3]. Later still, it was shown to be a substrate
for deoxyguanosine kinase, which is responsible for phosphorylation of purine nucleosides in
mitochondria [3]. Several mechanisms have been proposed by which cladribine can cause mitochondrial
damage and apoptotic cell death [4–8].
In contemporary medicine, cladribine is used in the treatment of lymphoid malignancies, most notably
for its efficacy against hairy cell leukemia [9]. Cladribine is also being evaluated against several other
indolent lymphoid malignancies, also in combination with other drug candidates [10,11].
The synthesis of cladribine has primarily relied on three major methods: (a) glycosylation reactions
of a nucleobase with a sugar [12–18], and its variations; (b) deoxygenation of the C2′ hydroxyl group of
a suitable nucleoside derivative [12,15,19,20]; (c) enzymatic glycosyl transfer reactions [21–24]; and (d)
conversion of readily available nucleoside precursors (some utilizing nucleosides for glycosyl transfer
reactions) [21,22,24–26]. Each of these methods has been used with varying levels of convenience and
success. Among the many approaches, one convenient method is the selective displacement of a leaving
group (chloride or aryl sulfonate) from the C6 position of a suitable purine nucleoside precursor. Despite
the availability of this selective S Ar displacement, we find that no other N6-substituted cladribine
N
analogues have been synthesized by such a method. Because of our interest in broadening the utilities of
6
O -(benzotriazol-1-yl)purine nucleoside derivatives, we elected to evaluate the synthesis of N6-substituted
cladribine analogues via amide-bond activation of guanine nucleosides with (benzotriazol-1yl-
oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

Molecules 2015, 20
. Results and Discussion
18439
2
For the modification of the nucleobases of inosine, and 2′-deoxyinosine (3 examples), BOP had been
used for in situ activation of the amide moieties in these substrates, followed by S Ar displacement with
N
amines [27,28]. It was proposed that reaction of the amide group with BOP proceeded via a phosphonium
intermediate, which could be directly captured by a reactive amine [27]. On the other hand, with less
reactive amines, the O-(benzotriazol-1-yl) intermediate can be formed by competitive capture of the
phosphonium intermediate by the benzotriazol-1-yloxy anion [27].
6
In 2007, we first reported the isolation of O -(benzotriazol-1-yl)inosine and -2′-deoxyinosine by
amide-bond activation with BOP, in the absence of a nucleophile [29], an observation that was later
reconfirmed by others [30,31]. These electrophilic nucleosides, which are stable to storage, are exceptionally
good partners in S
N
Ar reactions with oxygen, nitrogen, and sulfur nucleophiles [29]. Subsequently, we
6
demonstrated that O -(benzotriazol-1-yl)inosine and -2′-deoxyinosine can also be prepared via the use of
PPh
3
/I
2
and 1-hydroxybenzotriazole [32]. The amide-bond activation protocol was then modified to tether
6
the O -(benzotriazol-1-yl) nucleosides onto a polymer support for high-throughput type of applications [33].
6
Interestingly, the amide bond activation when applied to the urea functionality of O -benzyl-protected
2
2′-deoxyxanthosine did not yield the O -(benzotriazol-1-yl) derivative but rather terminated in an isolable
and synthetically useful phosphonium salt [34]. We also showed that guanosine and 2′-deoxyguanosine
6
undergo facile reactions with BOP, and that O -(benzotriazol-1-yl) guanine nucleosides are effective
substrates for S Ar reactions as well [35]. In the combined course of these investigations we had
N
ascertained plausible operative mechanisms of these amide-activation reactions, results that were later
applied to a one-pot etherification protocol for purine nucleosides and pyrimidines [36].
Considering only the nucleoside modification literature, our BOP-mediated amide-activation
methodology has found wide application [37–43]. Of specific interest to our current work was a
report wherein some of our results on guanosine derivatives were reevaluated [44]. In addition to
2′,3′,5′-tri-O-(t-butyldimethylsilyl)guanosine, which we had originally investigated, five other compounds
were also studied: unprotected 2′-deoxyguanosine, its 2′,3′,5′-triacetyl and the 2′,3′-isopropylidene
derivatives, and two 2′,3′-isopropylidiene 5′-carboxamides [44]. These products were subsequently
tested in diazotization-halogenation reactions, en route to 2-chloro and 2-iodo adenosine analogues [44].
2
,6-Dihalopurine ribonucleosides are more readily accessible for this purpose, as compared to 2′-deoxy
derivatives. Thus, we were intrigued by the fact that diazotization-halogenation reactions of
6
O -(benzotriazol-1yl)-2′-deoxyguanosine have not been reported. This prompted use to investigate the
chlorination and bromination of this compound, and in this context we decided to evaluate the synthesis
of cladribine and other cladribine analogues via such an approach.
In 2001, diazotization reactions leading to cladribine and other halo derivatives have been performed
on unprotected 2,6-diaminopurine 2′-deoxyribonucleoside [45]. However, no substituents other than NH
2
were introduced into the C6 position, and the synthesis of the precursor is not particularly convenient. For
the present study, we anticipated the need for saccharide protection, and both acetyl and t-butyldimethysilyl
(TBS) protecting groups were considered. Between these, TBS was selected because acetyl groups can be
susceptible to cleavage with amines, which could be a complicating problem in the method development
stage. Thus, nucleosides 1a and 1b were silylated to give the corresponding products 2a and 2b, which
6
were converted to the O -(benzotriazol-1-yl) guanosine derivatives 3a and 3b, respectively (Scheme 1).

Molecules 2015, 20
18440
6
Scheme 1. Preparation of O -(benzotriazol-1-yl) guanosine derivatives and the chlorination reaction.
We opted for diazotization-chlorination conditions using t-BuONO/TMSCl, a reagent combination
initially introduced for nucleoside modification in 2003 [46]. We [47] and others [44] have previously
used non-aqueous conditions for halogenation at the C2 position of purine nucleoside derivatives. Under
these conditions, reactions of substrate 3a proceeded modestly. However, the obtained product was
6
contaminated with ~30% of the C2 protio O -(benzotriazol-1-yl)-3′,5′-di-O-(t-butyldimethylsilyl)-2′-
+
−
deoxyinosine (Table 1, entries 1 and 2). With the combination of t-BuONO/TMSCl/(BnNEt ) Cl , no
3
C2 protio product was observed, but only a low product yield was obtained (entry 3).
6
Table 1. Evaluation of conditions for the diazotization/chlorination of O -(benzotriazol-1-
yl)-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyguanosine.
Entry Conditions
Scale
Yield
43% a
58% a
26%
1
2
3
4
5
6
7
8
9
t-BuONO (2.6 equiv), TMSCl (2.6 equiv), CH
t-BuONO (10 equiv), TMSCl (5 equiv), CH Cl
t-BuONO (10 equiv), TMSCl (0.4 equiv), (BnNEt
t-BuONO (3.5 equiv), SbCl (1.4 equiv), ClCH CH
t-BuONO (10 equiv), (BnNEt ) Cl (10 equiv), CH
t-BuONO (20 equiv), SbCl (0.4 equiv), (BnNEt
t-BuONO (10 equiv), (BnNEt
t-BuONO (3.5 equiv), SbCl (1.4 equiv), CH
t-BuONO (3.5 equiv), SbCl (1.4 equiv), CH
t-BuONO (3.5 equiv), SbCl (1.4 equiv), CH
t-BuONO (3.5 equiv), SbCl
t-BuONO (3.5 equiv), SbCl
2
Cl
2
, −10 °C to 0 °C, 2 h
73 μmol of 3a
73 μmol of 3a
49 μmol of 3a
49 μmol of 3a
49 μmol of 3a
49 μmol of 3a
2
2
, 0 °C, 1 h
+
−
3
) Cl (10 equiv), CH
Cl, −10 °C to −15 °C, 1.5 h
Cl , −78 °C and then rt, 1 h
2 2
Cl , −10 °C to 0 °C, 2 h
3
2
2
35%
+
−
3
2
2
39%
+
−
3
3
) Cl (20 equiv), CH
) Cl (10 equiv), (Me Si) NH (10 equiv), CH
Cl , −10 °C, 2 h
Cl
2
Cl
2
, −10 °C to 0 °C, 5 h
39%
+
−
3
3
2
2
Cl
2
, −10 °C to 0 °C, 4 h 49 μmol of 3a
35%
39% b
3
2
2
49 μmol of 3a
73 μmol of 3a
3
2
2
, −10 °C to −15 °C, 2 h
51%
1
0
1
2
3
3
2
2
Cl , −10 °C, 2 h
0.14 mmol of 3a 60%
1.6 mmol of 3a 65%
1
1
1
3
(1.4 equiv), CH
2
Cl
2
, −10 °C to −15 °C, 2 h
, −10 °C to −15 °C, 3.5 h
, 0 °C, 1 h
3
(1.4 equiv), CH
2
Cl
2
0.27 mmol of 3b 61%
t-BuONO (10 equiv), TMSCl (5 equiv), CH
2
Cl
2
2.69 mmol of 3b 68%
a
1
Yield was calculated on the basis of the molecular weight of compound 4a. However, by H-NMR
(
500 MHz, CDCl
3
), the chromatographically homogenous product band was observed to contain a 2:1 ratio of
6
compound 4a and the C2 protio O -(benzotriazol-1-yl)-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyinosine. In
b
addition to these, a minor uncharacterized nucleoside byproduct was also formed; Compound 3a dissolved in
2 2 2 2
CH Cl was added to the mixture of reagents in CH Cl .
These results compelled us to consider other conditions. SbCl
3
and SbBr have previously been used
3
for the halogenation of nucleosides [48,49] Thus, the next series of experiments involved SbCl
3
,

Molecules 2015, 20
18441
+
−
(BnNEt
3
) Cl , and combinations of these reagents (entries 4–11). Whereas most experiments yielded
only 35%–39% of product 4a, a reasonable yield improvement was observed in entry 9. It was noted that
efficient filtration of the reaction mixture after workup is critical to obtaining a good product recovery due
to the pasty nature of the mixture (see the Experimental Section). On larger scales, better product recoveries
were observed (entries 10 and 11). By comparison, diazotization/chlorination of the ribose derivative 3b
proceeded well with both t-BuONO/SbCl (entry 12) and t-BuONO/TMSCl (entry 13), with the latter
3
providing a better yield of compound 4b. No C2 protio product was apparent in the reactions of
precursor 3b.
The next stage in the chemistry involved S Ar reactions at the C6 positions of substrates 4a and 4b.
N
Cladribine and its ribose analogue were prepared by reactions with aqueous ammonia (see the Experimental
Section for details). In order to prepare other analogues, reactions were conducted with 1.5 equiv each of
methylamine, dimethylamine, pyrrolidine, piperidine, morpholine, and N,N,N′-trimethylethylenediamine
(products, reaction times, and yields are shown in Figure 1).
Figure 1. Structures of the products, reaction times, and yields obtained in the S Ar reactions.
N
Most reactions proceeded smoothly and in good to high yields. Methylamine (2 M in THF) was used
for the synthesis of 6a and 6b, whereas a 40% aqueous solution of dimethylamine was used for the
synthesis of 7a and 7b. As we have previously shown, water is not generally detrimental to reactions
of these benzotriazolyl purine nucleosides [35]. In reactions of 4a and 4b with N,N,N′-tri-
methylethylenediamine, yields were lower. In each ~10%–15% of compounds 7a and 7b were isolated as
byproducts. The source of dimethylamine is currently unknown but its origin can be linked to N,N,N′-
trimethylethylenediamine.
Finally, for biological testing, desilylation was performed. Because we did not anticipate decomposition
of starting materials or products, we only tested the use of KF (2 equiv/silyl group) in MeOH at 80 °C
(
Scheme 2). The results of the desilylation reactions are shown in Table 2.

Molecules 2015, 20
18442
Scheme 2. Desilylation of the protected nucleosides.
Table 2. Structures of the products, reaction times, and yields of the desilylated compounds.
Entry
R
Reaction Times/KF Equiv
24 h with 4 equiv KF
24 h with 6 equiv KF
24 h with 4 equiv KF
16 h with 6 equiv KF
24 h with 4 equiv KF
16 h with 6 equiv KF
Yields
1
2
3
4
5
6
12a: 72%
12b: 74%
13a: 85%
13b: 75%
14a: 82%
14b: 75%
7
8
20 h with 4 equiv KF
16 h with 6 equiv KF
15a: 81%
15b: 85%
9
24 h with 4 equiv KF
16 h with 6 equiv KF
16a: 71%
16b: 84%
1
1
1
0
1
2
26 h with 4 equiv KF
16 h with 6 equiv KF
17a: 74%
17b: 78%
1
1
3
4
24 h with 4 equiv KF
24 h with 6 equiv KF
18a: 84%
18b: 65%
Because there is currently no known method for the diazotization/bromination of compound 3a, we
investigated a route similar to that for chlorination. Results from these experiments are listed in Table 3.
What is notable with the diazotization/bromination, in contrast to the chlorination, was that use of
3
3
.5 equiv of t-BuONO led to incomplete conversion over 2 h at −10 to −15 °C. Addition of another
.5 equiv of t-BuONO then led to complete conversion over an additional 1 h.
A similar conversion of 3b led to the ribose analogue 19b in a comparable 64% yield (Table 3, entry 3).
Compound 19a was then converted to the bromo analogue of cladribine as shown in Scheme 3. S
displacement with aqueous NH proceeded in 83% yield producing compound 20a, which was finally
desilylated with KF in anhydrous MeOH at 80 °C (28 h) to yield 2-bromo-2-deoxyadenosine (21a) in
N
Ar
3
6
6% yield. Corresponding conversions of the ribose analog 19b, via intermediate 20b, gave compound
1b. Yields for these conversions were comparable to the deoxyribose series.
2

Molecules 2015, 20
18443
6
Table 3. Diazotization-bromination of silyl-protected O -(benzotriazol-1-yl) guanine nucleosides.
Entry
Conditions
Scale
Yield
1
2
3
t-BuONO (7.0 equiv), SbBr
t-BuONO (7.0 equiv), SbBr
t-BuONO (7.0 equiv), SbBr
3
3
3
(1.4 equiv), CH
(1.4 equiv), CH
(1.4 equiv), CH
2
2
2
Br
Br
Br
2
2
2
, −10 °C to −15 °C, 3 h
73 μmol of 3a
69%
, −10 °C to −15 °C, 3 h 0.49 mmol of 3a 63%
, −10 °C to −15 °C, 3 h 0.49 mmol of 3b 64%
Scheme 3. Synthesis of 2-bromo-2′-deoxyadenosine.
Notably, previously unknown compound 19a and 19b are relatively easily prepared, new bifunctional
reactive nucleosides that can undergo S Ar reactions at the C6 and metal-mediated reactions at the C2
N
position. To the extent that C-Cl bonds can be activated by metal catalysts compounds 4a and 4b also
offer this type of orthogonal reactivity. Results from the orthogonal reactivities of these new halo
nucleosides will be reported in the future.
Results of Tests against HCL, TCL, CLL, and MDA-MB-231 Breast Cancer Cells
The newly synthesized compounds as well as cladribine and its bromo analogue were tested against
HCL, TCL, and CLL. Data from these assays are shown in Table 4.
From these data, across the entire series, cladribine (12a) was best. The bromo analogue of cladribine
(
21a) showed lower activities. A comparison of the ribose analogue of cladribine (12b) and the bromo
analogue of cladribine (21a) is interesting. Both compounds 12b and 21a show comparable activities
against HCL, but the latter shows higher activities against TCL and CLL. The bromo ribose derivative

Molecules 2015, 20
18444
2
1b showed activity but was inferior to compounds 12a, 12b, and 21a. In this series, the only other
compound to demonstrate any activity was the piperidinyl derivative 16a.
Table 4. IC50 values (μM) of the cladribine and its analogues on HCL, TCL, and CLL a.
HCL
TCL
CLL
Compound
ATP 3H-Leu ATP 3H-Leu ATP 3H-Leu
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
21a
21b
0.065 0.090 0.020 0.039 0.004 0.011
0.81
>33
>32
>32
>30
>29
>28
11.5
>27
>28
>27
>27
>26
0.76
1.8
0.85
>33
>32
>32
>30
>29
>28
16.3
>27
>28
>27
>27
>26
0.96
7.2
4.81
>33
>32
>32
>30
>29
>28
9.6
9.40
>33
>32
>32
>30
>29
>28
9.0
1.33
>33
0.88
>33
>32
>32
>30
>29
>28
5.4
b
ND
>32
>30
>29
>28
4.3
b
b
>27
>28
>27
>27
>26
0.13
>27
>28
>27
>27
>26
0.16
10.2
ND
>28
>27
>27
>26
0.13
2.0
ND
>28
>27
>27
>26
0.14
4.0
8.6
a
b
One patient per disease type; ND = not determined.
The compounds were also tested against adenocarcinoma MDA-MB-231 breast cancer cells.
These cells were treated with three-fold serial dilutions of the compounds ranging from 0–1.8 mM for
2
4 h. Viable cells were fixed with cold methanol and nuclei were stained with 0.4% propidium iodide
(
PI). Cell viability was calculated as the percentage of surviving cells after treatment as measured by
differences in fluorescence units between treated and untreated wells (Figure 2).
IC50 values were obtained from dose response curve fittings using the non-linear regression function
®
of GraphPad Prism (La Jolla, CA, USA). Dashed horizontal line represents 50% cell viability. Columns
represent means ± SEM of at least three independent experiments. Significant differences are described
with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to control.
From the IC50 values shown in Table 5, the two compounds that emerged as most promising were
cladribine 12a and its ribose analogue 12b, followed by the bromo derivatives 21a and 21b, which were
about 10 times less active.
Table 5. IC50 values (mM) of the compounds synthesized on MDA-MB-231 breast cancer cells.
2
′-Deoxyribose Series
2a 13a 14a 15a 16a 17a 18a 21a 12b 13b 14b 15b 16b 17b 18b 21b
.05 2.15 6.28 2.54 4.18 2.32 1.88 0.64 0.06 2.39 1.70 1.54 2.45 2.57 2.91 0.55
Ribose Series
1
0

Molecules 2015, 20
18445
Figure 2. Effects of cladribine analogues on breast cancer cell viability. (A): Deoxyribose
series and (B): Ribose series. Significant differences are described with * p ≤ 0.05,
* p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to control.
*
3. Experimental Section
3.1. General Considerations
Thin-layer chromatography was performed on 200 μm aluminum-foil-backed silica gel plates for the
′-deoxynucleosides and on Merck 60F254 (Merck, Billerica, MA, USA) for the ribose analogues. Column
2
chromatographic purifications were performed on 100–200 mesh silica gel. CH
2
Cl for the chlorination
2
reactions was distilled over CaH . Precursors 3a and 3b were prepared as described previously [35]. The
2
yield of 3a was 71% on a 2.52 mmol scale and the yield of 3b was 71% on a 2.1 mmol scale. TMSCl was
redistilled prior to use and all other commercially available compounds were used without further
1
purification. H-NMR spectra were recorded at 500 MHz or at 400 MHz in the solvents indicated under
13
the individual compound headings and are referenced to residual protonated solvent resonances. C-NMR
spectra were recorded at 125 MHz or at 100 MHz in the solvents indicated under the individual compound
headings and are referenced to the solvent resonances (Supplementary Materials). Chemical shifts (δ)
are reported in parts per million (ppm), and coupling constants (J) are in hertz (Hz). Standard abbreviations
are used to designate resonance multiplicities (s = singlet, d = doublet, t = triplet, dd = doublet of doublet,
ddd = doublet of doublet of doublet, quint = quintet, m = multiplet, br = broad, app = apparent). The

Molecules 2015, 20
18446
saccharide carbons of the nucleoside are numbered 1′ through 5′ starting at the anomeric carbon atom and
proceeding via the carbon chain to the primary carbinol carbon atom. The purinyl proton is designated as
H-8 and the saccharide protons are designated on the basis of the carbon atom they are attached to.
6
O -(Benzotriazol-1-yl)-2-chloro-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (4a):
A mixture of compound 3a (1.0 g, 1.63 mmol) and SbCl
3
(520.6 mg, 2.28 mmol) in dry CH
Cl
2 2
(16.3 mL) was cooled to −15 °C using dry ice and acetone, in a nitrogen atmosphere. t-BuONO (0.678 mL,
5.70 mmol) was added dropwise and the mixture was stirred at −10 to −15 °C for 3.5 h, at which time
TLC indicated the reaction to be complete. The reaction mixture was poured into ice-cold, saturated
aqueous NaHCO (25 mL) with stirring. The mixture was filtered using vacuum (note: use of vacuum
for this filtration is critical for maximizing product recovery) and the residue was washed with CH
3
2
2
Cl
Cl
2
2
(
(
25 mL). The organic layer was separated and the aqueous layer was back extracted with CH
2 × 15 mL). The combined organic layer was washed with water (15 mL) and brine (15 mL), dried over
SO , and evaporated under reduced pressure. Purification of the crude material on a silica
gel column sequentially eluted with hexanes, 5% EtOAc in hexanes, followed by 20% EtOAc in hexanes
gave 0.67 g (65% yield) of compound 4a as a white foam. R (SiO and 30% EtOAc in hexanes) = 0.55.
1H-NMR (500 MHz, CDCl
): δ 8.47 (s, 1H, H-8), 8.09 (d, J = 8.3 Hz, 1H, Ar-H), 7.52–7.41 (m, 3H, Ar-H),
.45 (t, J = 5.8 Hz, 1H, H-1′), 4.62 (m, 1H, H-3′), 4.01 (m, 1H, H-4′), 3.89 (dd, J = 3.4, 11.2 Hz, 1H,
H-5′), 3.76 (dd, J = 2.4, 11.2 Hz, 1H, H-5′), 2.61 (app quint, Japp ~ 6.2 Hz, 1H, H-2′), 2.49–2.48 (app quint,
anhydrous Na
2
4
f
2
3
6
13
J
app ~ 5.8 Hz, 1H, H-2′), 0.88 (s, 18H, t-Bu), 0.08 and 0.07 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ):
3
δ 159.2, 154.9, 152.6, 144.4, 143.5, 129.0, 128.8, 125.0, 120.7, 119.1, 108.6, 88.4, 85.3, 71.6, 62.6, 41.6,
2
6
6.1, 25.9, 18.5, 18.1, −4.5, −4.7, −5.2, −5.3. HRMS (TOF) calcd for C28
H43ClN
7
O
4
Si
[M + H]+
2
32.2598, found 632.2583.
2-Chloro-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyadenosine (5a): To a solution of compound 4a
(126.0 mg, 0.2 mmol) in 1,2-DME (2 mL), 28%–30% aqueous ammonia (32 μL) was added, and the
mixture was stirred at room temperature for 1.5 h. The mixture was diluted with EtOAc (5 mL) and
washed with 5% aqueous NaCl (5 mL). The organic layer was separated and the aqueous layer was back
extracted with EtOAc (5 mL). The combined organic layer was dried over anhydrous Na
2
SO
4
and
evaporated under reduced pressure. The crude material was chromatographed on a silica gel column by
sequential elution with 20% EtOAc in hexanes followed by 5% MeOH in CH
2
Cl
2
to afford 85.0 mg
1
(
82% yield) of compound 5a as a white solid. R
500 MHz, CDCl ): δ 8.12 (s, 1H, H-8), 6.54 (br s, 2H, NH
H, H-3′), 3.99 (app q, Japp ~ 3.4 Hz, 1H, H-4′) 3.88 (dd, J = 3.9, 11.2 Hz, 1H, H-5′), 3.76 (dd, J = 3.0,
1.2 Hz, 1H, H-5′), 2.61 (app quint, Japp ~ 6.4 Hz, 1H, H-2′), 2.44–2.39 (ddd, J = 3.1, 6.3, 13.2 Hz, 1H,
f
(SiO
2
and 10% MeOH in CH
2
Cl
2
) = 0.40. H-NMR
(
3
2
), 6.38 (t, J = 6.1 Hz, 1H, H-1′), 4.61 (m,
1
1
1
3
H-2′), 0.90 (s, 18H, t-Bu), 0.09 and 0.08 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ): δ 156.2, 154.0,
3
1
50.4, 139.5, 118.7, 87.9, 84.5, 71.7, 62.6, 41.3, 25.9, 25.7, 18.4, 18.0, −4.7, −4.8, −5.4, −5.5. HRMS
+
(
TOF) calcd for C22
H40ClN
5
O
3
Si Na [M + Na] 536.2250, found 536.2252.
2
3
.2. General Procedure for the Synthesis of Cladribine Analogues
To a solution of compound 4a (126.0 mg, 0.2 mmol) in 1,2-DME (2 mL) the appropriate amine
(
1.5 equiv) was added, and the mixture was stirred at room temperature. The mixture was diluted with

Molecules 2015, 20
18447
EtOAc (5 mL for compounds 6a, 9a, 11a, and 15 mL for compounds 7a, 8a, 10a) and washed 5% aqueous
NaCl (5 mL for compounds 6a, 9a, 11a, and 15 mL for compounds 7a, 8a, 10a). The organic layer was
separated and the aqueous layer was back extracted with EtOAc (5 mL for compounds 6a, 9a, 11a, and
1
5 mL for compounds 7a, 8a, 10a). The combined organic layer was dried over anhydrous Na
2
SO and
4
evaporated under reduced pressure. The crude material was chromatographed on a silica gel column; see
individual compound headings for details.
6
2
-Chloro-N -methyl-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyadenosine (6a): Compound 6a (85.0 mg,
80% yield) was obtained as a white, foamy solid after chromatography on a silica gel column by sequential
elution with 5% EtOAc in hexanes and 15% EtOAc in hexanes. R
f
(SiO and 30% EtOAc in hexanes) =
2
1
0
.26. H-NMR (500 MHz, CDCl
3
): δ 8.02 (s, 1H, H-8), 6.37 (t, J = 6.4 Hz, 1H, H-1′), 6.17 (br s, 1H,
NH), 4.60 (m, 1H, H-3′), 3.97 (app q, Japp ~ 3.4 Hz, 1H, H-4′), 3.87 (dd, J = 4.1, 11.2 Hz, 1H, H-5′), 3.75
(
dd, J = 3.0, 11.2 Hz, 1H, H-5′), 2.61 (app quint, Japp ~ 6.4 Hz, 1H, H-2′), 2.42–2.37 (ddd, J = 2.8, 6.1,
1
3
1
2.7 Hz, 1H, H-2′), 0.90 (s, 18H, t-Bu), 0.09 and 0.08 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ):
3
δ 155.9, 154.5, 149.2; 138.7, 119.1, 87.9, 84.4, 71.8, 62.7, 41.2, 27.5, 25.9, 25.7, 18.4, 18.0, −4.7, −4.8,
+
−
5.4, −5.5. HRMS (TOF) calcd for C23
H42ClN
5
O
3
Si
2
Na [M + Na] 550.2407, found 550.2419.
6
6
2
-Chloro-N ,N -dimethyl-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyadenosine (7a): Compound 7a (91.1 mg,
8
4% yield) was obtained as a colorless, thick gum after chromatography on a silica gel column by
sequential elution with hexanes, 5% EtOAc in hexanes, and 20% EtOAc in hexanes. R
f
(SiO and 30%
2
1
EtOAc in hexanes) = 0.70. H-NMR (500 MHz, CDCl
3
): δ 7.93 (s, 1H, H-8), 6.36 (t, J = 6.3 Hz, 1H,
H-1′), 4.58 (m, 1H, H-3′), 3.95 (m, 1H, H-4′), 3.83 (dd, J = 4.4, 11.2 Hz, 1H, H-5′), 3.73 (dd, J = 2.9,
1
1
1
1.2 Hz, 1H, H-5′), 3.50 (br s, 6H, N(CH
3
) ), 2.58 (app quint, Japp ~ 6.5 Hz, 1H, H-2′), 2.39–2.35 (m,
2
1
3
H, H-2′), 0.88 (s, 18H, t-Bu), 0.08 and 0.06 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ): δ 155.2,
3
53.8, 151.4, 137.2, 119.6, 87.9, 84.4, 72.1, 62.9, 41.1, 26.1, 26.0, 25.9, 25.8, 18.6, 18.2, −4.5, −4.6, −5.2,
+
−
5.3. HRMS (TOF) calcd for C24H45ClN O
5 3
Si
2
[M + H] 542.2744, found 542.2749.
2
-Chloro-6-(pyrrolidin-1-yl)-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (8a):
Compound 8a (93.3 mg, 82% yield) was obtained as a colorless, thick gum after chromatography on a
silica gel column by sequential elution with hexanes, 5% EtOAc in hexanes, and 20% EtOAc in hexanes.
1
R
f
(SiO
2
and 30% EtOAc in hexanes) = 0.65. H-NMR (500 MHz, CDCl
3
): δ 7.92 (s, 1H,
H-8), 6.37 (t, J = 6.6 Hz, 1H, H-1′), 4.59 (m, 1H, H-3′), 4.13 (br s, 2H, pyrrolidinyl NCH), 3.96 (m, 1H,
H-4′), 3.83 (dd, J = 4.9, 11.2 Hz, 1H, H-5′), 3.74 (dd, J = 3.9, 11.2 Hz, 1H, H-5′), 3.79–3.73 (br m, 2H,
pyrrolidinyl NCH), 2.61 (app quint, Japp ~ 6.5 Hz, 1H, H-2′), 2.39–2.35 (m, 1H, H-2′), 2.10–2.0 (br m, 2H,
pyrrolidinyl CH), 1.96–1.85 (br m, 2H, pyrrolidinyl CH), 0.90 (s, 18H, t-Bu), 0.09 and 0.07 (2s, 12H,
1
3
SiCH
3
). C-NMR (125 MHz, CDCl ): δ 154.3, 153.5, 151.0, 137.8, 119.8, 88.1, 84.4, 72.3, 63.1, 49.1,
3
4
1.08, 26.4, 26.2, 25.9, 24.3, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3. HRMS (TOF) calcd. for
+
C
26
H47ClN
5
O
3
Si [M + H] 568.2900, found 568.2906.
2
2
-Chloro-6-(piperidin-1-yl)-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (9a):
Compound 9a (99.0 mg, 85% yield) was obtained as a white, foamy solid after chromatography on a
silica gel column by sequential elution with 5% EtOAc in hexanes and 15% EtOAc in hexanes. R
f
(SiO
2
1
3
and 30% EtOAc in hexanes) = 0.65. H-NMR (500 MHz, CDCl ): δ 7.92 (s, 1H, H-8), 6.37 (t,

Molecules 2015, 20
18448
J = 6.6 Hz, 1H, H-1′), 4.59 (m, 1H, H-3′), 4.45–3.73 (br m, 4H, piperidinyl N(CH
2
)
2
), 3.96 (app q, Japp
~
3.6 Hz, 1H, H-4′), 3.83 (dd, J = 4.9, 11.2 Hz, 1H, H-5′), 3.74 (dd, J = 3.4, 11.2 Hz, 1H, H-5′), 2.59
(
app quint, Japp ~ 6.3 Hz, 1H, H-2′), 2.40–2.35 (ddd, J = 3.3, 6.2, 13.3 Hz, 1H, H-2′), 1.68 (br m, 6H,
1
3
piperidinyl CH), 0.90 (s, 18H, t-Bu), 0.09 and 0.07 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ): δ
3
1
53.8, 151.4, 136.7, 119.0, 109.9, 87.8, 84.2, 72.0, 62.8, 46.3, 40.9, 26.1, 25.9, 25.7, 24.6, 18.4, 18.0,
+
−
4.7, −4.8, −5.4, −5.5. HRMS (TOF) calcd for C27
H49ClN
5
O
3
Si [M + H] 582.3057 found 582.3074.
2
2-Chloro-6-(morpholin-4-yl)-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (10a):
Compound 10a (97.4 mg, 83% yield) was obtained as a colorless, thick gum after chromatography on a
silica gel column by sequential elution with hexanes, 5% EtOAc in hexanes, and 20% EtOAc in hexanes.
1
R
f
(SiO
2
and 30% EtOAc in hexanes) = 0.68. H-NMR (500 MHz, CDCl
3
): δ 7.98 (s, 1H, H-8), 6.37 (t, J
), 3.96 (m, 1H, H-
OCH ), 3.74 (dd, J =
.9, 11.2 Hz, 1H, H-5′), 2.56 (app quint, Japp ~ 6.5 Hz, 1H, H-2′), 2.40–2.36 (m, 1H,
=
6.3 Hz, 1H, H-1′), 4.58 (m, 1H, H-3′), 4.55–3.98 (br m, 4H, morpholinyl N(CH
)
2 2
4
′), 3.84 (dd, J = 4.4, 11.2 Hz, 1H, H-5′), 3.78 (t, J = 4.6 Hz, 4H, morpholinyl CH
2
2
2
1
3
H-2′), 0.89 (s, 18H, t-Bu), 0.08 and 0.07 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ): δ 154.0, 153.9,
3
1
51.8, 137.6, 119.3, 88.0, 84.4, 72.0, 67.1, 62.9, 43.5 (br), 41.3, 26.1, 26.0, 25.9, 18.6, 18.2, −4.5, −4.6,
+
−
5.2, −5.3. HRMS (TOF) calcd for C26
H47ClN
5
O
4
Si [M + H] 584.2850, found 584.2855.
2
2
-Chloro-6-[(2-dimethylamino)ethyl)(methyl)amino)]-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-
ribofuranosyl]purine (11a): Compound 11a (77.0 mg, 64% yield) was obtained as a pale-yellow, thick
gum after chromatography on a silica gel column by sequential elution with 10% EtOAc in hexanes,
2
0% EtOAc in hexanes, and 10% MeOH in CH
1H-NMR (500 MHz, CDCl
): δ 7.94 (s, 1H, H-8), 6.36 (t, J = 6.3 Hz, 1H, H-1′), 4.62 (m, 1H, H-3′),
.28–3.84 (br m, 2H, NCH ), 3.99 (app q, Japp ~ 3.7 Hz, 1H, H-4′), 3.85 (dd, J = 4.4, 11.2 Hz, 1H,
H-5′), 3.78 (dd, J = 3.4, 11.2 Hz, 1H, H-5′), 3.47 (br s, 3H, NCH ), 2.65–2.59 (m, 2H, NCH and 1H, H-
′), 2.41–2.37 (ddd, J = 4.2, 6.2, 13.1 Hz, 1H, H-2′), 2.34 (s, 6H, N(CH ), 0.92 and 0.91 (s, 18H,
): δ 154.9, 153.8, 151.4, 137.2,
2
Cl
2
. R
f
(SiO
2
and 10% MeOH in CH
2
Cl
2
) = 0.10.
3
4
2
3
2
2
)
3 2
13
t-Bu), 0.11, 0.083, and 0.079 (3s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl
3
1
19.4, 87.9, 84.4, 72.0, 62.9, 57.0, 48.6, 45.7, 41.0, 36.9, 26.0, 25.8, 18.4, 8.0, −4.6, −4.8, −5.4, −5.5.
+
HRMS (TOF) calcd for C27
H52ClN
6
O
3
Si [M + H] 599.3322 found 599.3313.
2
6
O -(Benzotriazol-1-yl)-2-chloro-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (4b): To a
solution of t-BuONO (2.78 g, 26.91 mmol) in CH
°C. To this mixture a solution of 3b (2 g, 2.69 mmol) in CH
then the reaction mixture was stirred at 0 °C for 1 h at which time the reaction was completed as indicated
by TLC. The reaction mixture was diluted with CH Cl (200 mL), washed with saturated NaHCO
100 mL), H O (100 mL), and brine (100 mL). The organic layer was dried over anhydrous Na SO and
Cl
2 2
(197 mL) was added TMSCl (1.46 g, 13.45 mmol) at
0
2
Cl (197 mL) was added dropwise, and
2
2
2
3
(
2
2
4
evaporated under reduced pressure. Purification of the crude material on a silica gel column sequentially
eluted with hexanes, 5% EtOAc in hexanes, and 20% EtOAc in hexanes gave 1.4 g (68% yield) of
1
compound 4b as a white foam. R
CDCl
H, H-1′), 4.57 (t, J = 4.2 Hz, 1H, H-2′), 4.34 (t, J = 4.4 Hz, 1H, H-3′), 4.18–4.15 (m, 1H, H-4′), 4.08
dd, J = 3.6, 11.6 Hz, 1H, H-5′), 3.83 (dd, J = 2.4, 11.6 Hz, 1H, H-5′), 0.96, 0.93, and 0.88 (3s, 27H,
f
(SiO
2
and 30% EtOAc in hexanes) = 0.55. H-NMR (400 MHz,
3
): δ 8.56 (s, 1H, H-8), 8.14 (d, J = 8.0 Hz, 1H, Ar-H), 7.55–7.45 (m, 3H, Ar-H), 6.05 (d, J = 4.0 Hz,
1
(

Molecules 2015, 20
18449
13
t-Bu), 0.15, 0.11, 0.02, and −0.11 (4s, 18H, SiCH
3
). C-NMR (100 MHz, CDCl
3
): δ 159.0, 154.9, 152.6,
1
1
44.4, 143.4, 128.9, 128.7, 124.9, 120.6, 119.0, 108.5, 89.4, 85.3, 71.2, 62.0, 26.1, 25.8, 25.6, 18.5, 18.0,
+
7.9, −4.3, −4.6, −4.7, −4.9, −5.3, −5.4. HRMS (TOF) calcd for C34
H57ClN
7
O
5
Si [M + H] 762.3412,
3
found 762.3427.
2
-Chloro-2′,3′,5′-tri-O-(t-butyldimethylsilyl)adenosine (5b): To a solution of compound 4b (500 mg,
.655 mmol) in 1,2-DME (8 mL), 28%–30% aqueous ammonia (64 μL) was added, and the mixture was
0
stirred at room temperature for 1.5 h. The reaction mixture was diluted with EtOAc (25 mL) and washed
% aqueous NaCl (25 mL). The organic layer was separated and the aqueous layer was back extracted
with EtOAc (25 mL). The combined organic layer was dried over anhydrous Na SO and evaporated
5
2
4
under reduced pressure. The crude material was chromatographed on a silica gel column by sequential
elution with 20% EtOAc in hexanes and 5% MeOH in EtOAc to afford 310 mg (75% yield) of compound
1
5
b as an off-white solid. R
s, 1H, H-8), 6.17 (br s, 2H, NH
J = 4.6 Hz, H-3′), 4.14–4.06 (m, 1H, H-4′), 4.06 (dd, J = 4.8, 11.6 Hz, 1H, H-5′), 3.80 (dd, J = 2.8, 11.2 Hz,
f
(SiO
2
and 10% MeOH in EtOAc) = 0.21. H-NMR (400 MHz, CDCl
3
): δ 8.12
(
2
), 5.93 (d, J = 4.8 Hz, 1H, H-1′), 4.69 (t, J = 4.6 Hz, 1H, H-2′), 4.32 (t,
1
3
1
H, H-5′), 0.94, 0.91, and 0.84 (3s, 27H, t-Bu), 0.14, 0.09, −0.01, and −0.17 (4s, 18H, SiCH
3
). C-NMR
(
100 MHz, CDCl ): δ 156.1, 154.0, 150.7, 140.2, 118.9, 88.9, 85.4, 75.5, 71.7, 62.3, 26.0, 25.8, 25.7,
3
1
6
8.5, 18.0, 17.9, −4.3, −4.5, −4.7, −5.0, −5.3, −5.4. HRMS (TOF) calcd. for C28
H55ClN
5
O
4
Si
[M + H]+
3
44.3245 found 644.3217.
3
.3. General Procedure for the Synthesis of Ribose Analogues of Cladribine
To a solution of compound 4b (500 mg, 0.655 mmol) in 1,2-DME (8 mL) was added the appropriate
amine (1.5 equiv) and the mixture was stirred at room temperature. The mixture was diluted with EtOAc
25 mL) and washed with 5% aqueous NaCl (15 mL). The organic layer was separated and the aqueous
layer was back extracted with EtOAc (15 mL). The combined organic layers were dried over anhydrous
Na SO and evaporated under reduced pressure. The crude material was chromatographed on a silica gel
(
2
4
column. See individual compound headings for details.
6
2
-Chloro-N -methyl-2′,3′,5′-tri-O-(t-butyldimethylsilyl)adenosine (6b): Compound 6b (340 mg, 80% yield)
was obtained as a pale-yellow, thick gum after chromatography on a silica gel column by sequential
elution with 10% EtOAc in hexanes, 20% EtOAc in hexanes, and 50% EtOAc in hexanes. R
f
(SiO and
2
1
3
EtOAc) = 0.20. H-NMR (400 MHz, CDCl ): δ 8.02 (s, 1H, H-8), 6.04 (br s, 1H, NH), 5.91 (d, J = 5.2 Hz,
1
H, H-1′), 4.72 (t, J = 4.6 Hz, 1H, H-2′), 4.32 (t, J = 4.0 Hz, 1H, H-3′), 4.12–4.10 (m, 1H, H-4′), 4.06
dd, J = 5.2, 11.6 Hz, 1H, H-5′), 3.79 (dd, J = 2.8, 11.2 Hz, 1H, H-5′), 3.17 (s, 3H, CH ), 0.94, 0.92, and
). C-NMR (100 MHz, CDCl ): δ 155.9,
(
3
13
0.81 (3s, 27H, t-Bu), 0.13, 0.06, −0.02, and −0.19 (4s, 18H, SiCH
3
3
1
54.5, 139.3, 119.3, 88.8, 85.4, 75.3, 71.5, 65.2, 62.4, 26.0, 25.8, 25.7, 18.4, 18.0, 17.8, 14.0, 11.0, −4.4,
+
−
4.7, −5.1, −5.4, −5.7. HRMS (TOF) calcd for C29
H57ClN
5
O
4
Si
3
[M + H] 658.3401, found 658.3416.
6
6
2
-Chloro-N ,N -dimethyl- 2′,3′,5′-tri-O-(t-butyldimethylsilyl)adenosine (7b): Compound 7b (360 mg,
8
1% yield) was obtained as a pale-yellow, thick gum after chromatography on a silica gel column by
sequential elution with 10% EtOAc in hexanes and 20% EtOAc in hexanes. R
f
(SiO and 20% EtOAc in
2
1
hexanes) = 0.40. H-NMR (400 MHz, CDCl
3
): δ 7.93 (s, 1H, H-8), 5.90 (d, J = 5.2 Hz, 1H, H-1′), 4.77

Molecules 2015, 20
18450
(
t, J = 4.8 Hz, 1H, H-2′), 4.31 (t, J = 3.8 Hz, 1H, H-3′), 4.04–4.09 (m, 1H, H-4′), 4.06 (dd, J = 5.6,
1
2
1
1.2 Hz, 1H, H-5′), 3.78 (dd, J = 2.8, 11.2 Hz, 1H, H-5′), 3.58 (br s, 6H, CH
3
), 0.94, 0.92, and 0.82 (3s,
1
3
7H, t-Bu), 0.13, 0.10, −0.02, and −0.18 (4s, 18H, SiCH
3
). C-NMR (100 MHz, CDCl ): δ 155.9, 153.7,
3
51.4, 137.9, 119.7, 88.8, 85.4, 74.8, 72.0, 62.5, 38.4, 26.0, 25.8, 25.7, 18.4, 18.0, 17.9, −4.3, −4.7, −5.0,
+
−
5.4. HRMS (TOF) calcd for C30
H59ClN
5
O
4
Si
3
[M + H] 672.3558, found 672.3561.
2-Chloro-6-(pyrrolidin-1-yl)-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (8b): Compound
8
b (390 mg, 85% yield) was obtained as a pale-yellow, thick gum after chromatography on a silica gel
column by sequential elution with 10% EtOAc in hexanes and 20% EtOAc in hexanes. R
f
(SiO and 20%
2
1
EtOAc in hexanes) = 0.30. H-NMR (400 MHz, CDCl
3
): δ 7.90 (s, 1H, H-8), 5.90 (d, J = 5.2 Hz, 1H,
H-1′), 4.81 (t, J = 5.0 Hz, 1H, H-2′), 4.32 (t, J = 4.0 Hz, 1H, H-3′), 4.14–4.08 (m, 1H, H-4′ and br m,
H, pyrrolidinyl NCH), 4.06 (dd, J = 5.6, 11.2 Hz, 1H, H-5′), 3.77–3.73 (m, 3H, H-5′ and pyrrolidinyl
NCH), 2.04 (br m, 2H, pyrrolidinyl CH), 1.98 (br m, 2H, pyrrolidinyl CH), 0.93, 0.89, and 0.81 (3s,
2
1
3
2
7H, t-Bu), 0.12, 0.11, −0.03, and −0.20 (4s, 18H, SiCH
3
). C-NMR (100 MHz, CDCl ): δ 154.0, 153.4,
3
1
51.0, 138.0, 119.9, 88.7, 85.5, 74.6, 72.2, 62.6, 48.9, 47.6, 26.0, 25.8, 25.7, 18.4, 18.1, 17.9, −4.4, −4.6,
+
−
4.7, −5.0, −5.3. HRMS (TOF) calcd for C32
H61ClN
5
O
4
Si
3
[M + H] 698.3714 found 698.3731.
2-Chloro-6-(piperidin-1-yl)-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (9b): Compound
9b (375 mg, 80% yield) was obtained as a pale-yellow, thick gum after chromatography on a silica gel
column by sequential elution with 10% EtOAc in hexanes and 20% EtOAc in hexanes. R
f
(SiO and 20%
2
1
EtOAc in hexanes) = 0.50. H-NMR (400 MHz, CDCl
3
): δ 7.91 (s, 1H, H-8), 5.89 (d, J = 5.6 Hz, 1H,
),
.11–4.09 (m, 1H, H-4′), 4.06 (dd, J = 5.2, 10.8 Hz, 1H, H-5′), 3.77 (dd, J = 2.8, 10.8 Hz, 1H, H-5′),
.69 (br m, 6H, piperidinyl CH ), 0.94, 0.90, and 0.84 (3s, 27H, t-Bu), 0.12, 0.10, −0.02, and −0.17 (4s,
). C-NMR (100 MHz, CDCl ): δ 153.9, 153.8, 151.6, 137.6, 119.4, 88.8, 85.3, 74.7, 71.9,
H-1′), 4.79 (t, J = 4.8 Hz, 1H, H-2′), 4.32 (t, J = 4.0 Hz, 1H, H-3′), 4.22 (br s, 4H, piperidinyl NCH
2
4
1
1
6
2
1
3
8H, SiCH
3
3
2.5, 29.6, 25.8, 25.7, 24.6, 22.6, 18.4, 18.0, 17.9, 14.0, −4.3, −4.7, −5.0, −5.3. HRMS (TOF) calcd for
+
C
33
H63ClN
5
O
4
Si
3
[M + H] 712.3871, found 712.3875.
2-Chloro-6-(morpholin-4-yl)-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (10b): Compound
1
0b (430 mg, 90% yield) was obtained as a pale-yellow, thick gum after chromatography on a silica gel
column by sequential elution with 10% EtOAc in hexanes and 20% EtOAc in hexanes. R
f
(SiO and 20%
2
1
EtOAc in hexanes) = 0.47. H-NMR (400 MHz, CDCl
3
): δ 7.98 (s, 1H, H-8), 5.92 (d, J = 4.8 Hz, 1H, H-1′),
),
.12–4.09 (m, 1H, H-4′), 4.06 (dd, J = 5.2, 11.2 Hz, 1H, H-5′), 3.83 (t, J = 5.0 Hz, 4H, morpholinyl
OCH ), 3.77 (dd, J = 3.2, 11.2 Hz, 1H, H-5′), 0.94, 0.92, and 0.83 (3s, 27H, t-Bu), 0.13, 0.10, −0.01,
and −0.16 (4s, 18H, SiCH
4
4
.73 (t, J = 4.8 Hz, 1H, H-2′), 4.31–4.20 (t, J = 4.2 Hz, 1H, H-3′and br m, 4H, morpholinyl N(CH
)
2 2
CH
2
2
1
3
3
). C-NMR (100 MHz, CDCl
3
): δ 153.9, 153.7, 151.8, 138.1, 119.4, 88.8,
8
5.3,77.3,77.0,76.6,75.0, 71.8, 66.9, 62.3, 45.60, 29.6, 29.3, 26.0,25.8, 25.7, 22.6, 18.4, 18.0, 17.8,14.0,
+
−
4.3, −4.7, −5.0, −5.4. HRMS (TOF) calcd for C32
H61ClN
5
O
5
Si [M + H] 714.3664 found 714.3668.
3
2-Chloro-6-[(2-dimethylamino)ethyl)(methyl)amino)]-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]
purine (11b): Compound 11b (280 mg, 58%) was obtained as a pale-yellow, thick gum after
chromatography on a silica gel column by sequential elution with 10% MeOH in CH
2
Cl , 20% MeOH
2
1
in CH
2
Cl
2
, and 30% MeOH in CH
2
Cl
2
. R
f
(SiO
2
and 10% MeOH in CH
2
Cl
2
) = 0.10. H-NMR (500 MHz,

Molecules 2015, 20
18451
CDCl
H, H-3′), 4.15–4.10 (m, 1H, H-4′ and br s, 2H, NCH
J = 2.8, 11.2 Hz, 1H, H-5′ and br m, 2H, NCH ), 2.59 (br s, 2H, NCH
.92, and 0.82 (3s, 27H, t-Bu), 0.13, 0.10, −0.01, and −0.15 (4s, 18H, SiCH
3
): δ 7.97 (s, 1H, H-8), 5.90 (d, J = 4.8 Hz, 1H, H-1′), 4.72 (t, J = 4.8 Hz, 1H, H-2′), 4.32 (t, J = 3.8 Hz,
), 4.07 (dd, J = 5.2, 11.2 Hz, 1H, H-5′), 3.78 (dd,
), 2.32 (s, 6H, N(CH ), 0.94,
). C-NMR (100 MHz, CDCl ):
1
2
2
2
)
3 2
13
0
3
3
δ 154.8, 153.7, 151.4, 137.9, 119.5, 88.8, 85.2, 75.0, 71.9, 62.4, 45.7, 29.6, 26.0, 25.8, 25.7, 18.4, 18.0, 17.9,
+
−4.3, −4.7, −5.0, −5.4. HRMS (TOF) calcd for C33H66ClN O Si
6 4 3
[M + H] 729.4136, found 729.4157.
3.4. General Procedure for the Desilylation of Cladribine Analogues
To a 0.1 M solution of the silylated compound in anhydrous MeOH, KF (2 equiv/silyl group) was
added. The mixture was heated at 80 °C for 20–26 h, cooled, and silica gel was added. The mixture was
evaporated to dryness and the compound-impregnated silica gel was loaded onto a wet-packed silica gel
column. The products were obtained by elution with appropriate solvents (see the individual compound
headings for details).
2
-Chloro-2′-deoxyadenosine (12a): Prepared from compound 5a (60.0 mg, 0.117 mmol) and KF
(
27.0 mg, 0.467 mmol) in MeOH (1.17 mL). Chromatography on a silica gel column sequentially eluted
with 5% MeOH in EtOAc and 10% MeOH in EtOAc gave 24.0 mg (72% yield) of compound 12a as an
1
off-white solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.13. H-NMR (500 MHz, CD
3
OD): δ 8.28 (s,
1
1
2
1
H, H-8), 6.36 (t, J = 6.8 Hz, 1H, H-1′), 4.57 (m, 1H, H-3′), 4.04 (m, 1H, H-4′), 3.84 (dd, J = 2.9,
2.2 Hz, 1H, H-5′), 3.74 (dd, J = 3.4, 12.2 Hz, 1H, H-5′), 2.76 (app quint, Japp ~ 6.7 Hz, 1H, H-2′),
1
3
.43–2.39 (ddd, J = 2.9, 5.9, 13.2 Hz, 1H, H-2′). C-NMR (125 MHz, CD
3
OD): δ 158.3, 155.3, 151.4,
+
41.9, 119.8, 89.9, 87.0, 73.0, 63.6, 41.6. HRMS (TOF) calcd for C10
H12ClN
5
O
3
Na [M + Na] 308.0521,
found 308.0523.
6
2
-Chloro-N -methyl-2′-deoxyadenosine (13a) [50]: Prepared from compound 6a (80.0 mg, 0.154 mmol)
and KF (36.0 mg, 0.618 mmol) in MeOH (1.54 mL). Chromatography on a silica gel column sequentially
eluted with 2.5% MeOH in EtOAc and 5% MeOH in EtOAc gave 39.0 mg (85% yield) of compound
1
1
3a as a white, foamy solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.21. H-NMR (500 MHz, CD
3
OD): δ
8
.18 (s, 1H, H-8), 6.34 (t, J = 6.8 Hz, 1H, H-1′), 4.56 (m, 1H, H-3′), 4.04 (m, 1H, H-4′), 3.84 (dd,
), 2.76 (app quint,
app ~ 6.8 Hz, 1H, H-2′), 2.42–2.38 (ddd, J = 2.9, 5.8, 13.7 Hz, 1H, H-2′). C-NMR (125 MHz, CD OD):
J = 2.4, 12.2 Hz, 1H, H-5′), 3.74 (dd, J = 3.4, 12.2 Hz, 1H, H-5′), 3.05 (br s, 3H, NCH
3
1
3
J
3
δ 157.3, 155.5, 150.1, 141.2, 120.4, 89.9, 87.0, 73.0, 63.7, 41.6, 27.8. HRMS (TOF) calcd for
+
C
11
H14ClN
5
O
3
Na [M + Na] 322.0677, found 322.0682.
6
6
2
-Chloro-N ,N -dimethyl-2′-deoxyadenosine (Cladribine, 14a): Prepared from compound 7a (80.0 mg,
0.147 mmol) and KF (34.3 mg, 0.590 mmol) in MeOH (1.4 mL). Chromatography on a silica gel column
sequentially eluted with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave 38.0 mg (82%
yield) of compound 14a as a white, foamy solid. R (SiO and 5% MeOH in EtOAc) = 0.29.
1H-NMR (500 MHz, CD
OD): δ 8.16 (s, 1H, H-8), 6.35 (t, J = 6.8 Hz, 1H, H-1′), 4.56 (m, 1H, H-3′),
.04 (m, 1H, H-4′), 3.84 (dd, J = 2.9, 12.2 Hz, 1H, H-5′), 3.73 (dd, J = 3.4, 12.2 Hz, 1H, H-5′),
.70–3.10 (br m, 6H, N(CH ), 2.74 (app quint, Japp ~ 6.8 Hz, 1H, H-2′), 2.41–2.36 (ddd, J = 2.9, 5.8,
3.2 Hz, 1H, H-2′). 13C-NMR (125 MHz, CD
OD): δ 156.4, 154.6, 152.2, 140.0, 120.72, 89.8,
f
2
3
4
3
1
)
3 2
3

Molecules 2015, 20
18452
Na [M + Na] 336.0834,
+
8
6.9, 73.0, 63.7, 41.5, 39.0 (br). HRMS (TOF) calcd for C12
H16ClN
5
O
3
found 336.0823.
2
-Chloro-6-(pyrrolidin-1-yl)-9-(2-deoxy-β-D-ribofuranosyl)purine (15a): Prepared from compound 8a
60.0 mg, 0.105 mmol) and KF (24.5 mg, 0.422 mmol) in MeOH (1.0 mL). Chromatography on a silica
gel column sequentially eluted with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave
9.1 mg (81% yield) of compound 15a as a white solid. R (SiO and 5% MeOH in EtOAc) = 0.19.
1H-NMR (500 MHz, CD
OD): δ 8.19 (s, 1H, H-8), 6.36 (t, J = 7.1 Hz, 1H, H-1′), 4.59 (m, 1H, H-3′),
.14–4.07 (br m, 2H, pyrrolidinyl NCH), 4.04 (m, 1H, H-4′), 3.84 (dd, J = 3.4, 12.7 Hz, 1H, H-5′), 3.74
dd, J = 3.4, 12.2 Hz, 1H, H-5′), 3.71–3.62 (br m, 2H, pyrrolidinyl NCH), 2.75 (app quint, Japp ~ 6.7 Hz,
(
2
f
2
3
4
(
1
2
1
H, H-2′), 2.40–2.36 (ddd, J = 2.9, 5.8, 13.2 Hz, 1H, H-2′), 2.15–2.06 (br m, 2H, pyrrolidinyl CH),
1
3
.04–1.92 (br m, 2H, pyrrolidinyl CH). C-NMR (125 MHz, CD OD): δ 155.0, 154.7, 151.7, 140.6,
3
20.8, 89.9, 87.0, 73.0, 63.7, 50.2, 49.6, 41.6, 27.3, 25.2. HRMS (TOF) calcd for C14
H19ClN
5
O
3
+
[M + H] 340.1171, found 340.1149.
2
-Chloro-6-(piperidin-1-yl)-9-(2-deoxy-β-D-ribofuranosyl)purine (16a): Prepared from compound 9a
(
70.0 mg, 0.120 mmol) and KF (28.0 mg, 0.481 mmol) in MeOH (1.20 mL). Chromatography on a silica
gel column sequentially eluted with EtOAc and 2% MeOH in EtOAc gave 30.0 mg (71% yield) of
1
compound 16a as a white, foamy solid. R
CD
N(CH
H-5′), 2.74 (app quint, Japp ~ 6.8 Hz, 1H, H-2′), 2.40–2.35 (ddd, J = 2.7, 5.9, 13.5 Hz, 1H, H-2′), 1.74
f
(SiO
2
and 10% MeOH in EtOAc) = 0.46. H-NMR (500 MHz,
3
OD): δ 8.15 (s, 1H, H-8), 6.34 (t, J = 6.6 Hz, 1H, H-1′), 4.55 (m, 1H, H-3′), 4.18 (br s, 4H, piperidinyl
), 4.03 (m, 1H, H-4′), 3.83 (dd, J = 2.4, 12.2 Hz, 1H, H-5′), 3.73 (dd, J = 3.4, 12.2 Hz, 1H,
)
2 2
1
3
(
br m, 2H, piperidinyl CH
2
), 1.65 (br m, 4H, piperidinyl CH
2
). C-NMR (125 MHz, CD OD): δ 155.3,
3
1
54.9, 152.7, 139.6, 120.5, 89.8, 86.8, 73.0, 63.7, 47.8, 41.6, 27.3, 25.7. HRMS (TOF) calcd for
+
C
15
H20ClN
5
3
O Na [M + Na] 376.1147, found 376.1148.
2
-Chloro-6-(morpholin-4-yl)-9-(2-deoxy-β-D-ribofuranosyl)purine (17a): Prepared from compound 10a
80.0 mg, 0.137 mmol) and KF (31.8 mg, 0.548 mmol) in MeOH (1.4 mL). Chromatography on a silica
gel column sequentially eluted with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave
(
3
0
6.1 mg (74% yield) of compound 17a as a white, foamy solid. R
f
(SiO and 5% MeOH in EtOAc) =
2
1
.21. H-NMR (500 MHz, CD
3
OD): δ 8.22 (s, 1H, H-8), 6.37 (t, J = 7.1 Hz, 1H, H-1′), 4.56 (m, 1H,
), 4.04 (m, 1H, H-4′), 3.83 (dd, J = 2.9, 12.2 Hz, 1H,
OCH ), 3.74 (dd, J = 3.4, 12.2 Hz, 1H, H-5′), 2.74 (app
quint, Japp ~ 6.7 Hz, 1H, H-2′), 2.41–2.37 (ddd, J = 2.9, 5.8, 13.2 Hz, 1H, H-2′). C-NMR (125 MHz,
H-3′), 4.40–4.12 (br m, 4H, morpholinyl N(CH
)
2 2
H-5′), 3.79 (t, J = 4.9 Hz, 4H, morpholinyl CH
2
2
1
3
CD
3
OD): δ 155.2, 154.7, 152.7, 140.3, 120.6, 89.8, 86.8, 72.9, 68.0, 63.6, 47.0 (br), 41.5. HRMS (TOF)
+
calcd for C14
H19ClN
5
O
4
[M + H] 356.1120, found 356.1107.
2-Chloro-6-[(2-dimethylamino)ethyl)(methyl)amino)]-9-(2-deoxy-β-D-ribofuranosyl)purine (18a): Prepared
from compound 11a (70.0 mg, 0.117 mmol) and KF (27.0 mg, 0.467 mmol) in MeOH (1.17 mL).
Chromatography on a silica gel column sequentially eluted with 10% MeOH in EtOAc and 20% MeOH
in EtOAc gave 36.0 mg (84% yield) of compound 18a as a white, foamy solid. R
f
(SiO and 10% MeOH
2
1
in EtOAc) = 0.10. H-NMR (500 MHz, CD
3
OD): δ 8.14 (s, 1H, H-8), 6.35 (t, J = 6.9 Hz, 1H, H-1′), 4.56
(
m, 1H, H-3′), 4.12 (br s, 2H, NCH
2
), 4.03 (m, 1H, H-4′), 3.83 (dd, J = 3.4, 12.2 Hz, 1H, H-5′), 3.74 (dd,

Molecules 2015, 20
18453
J = 3.9, 12.2 Hz, 1H, H-5′), 3.48 (br s, 3H, NCH
3
), 2.76–2.71 (br m, 3H, NCH
2
and 1H, H-2′), 2.42–2.41
13
(
m, 1H, H-2′), 2.39 (s, 6H, N(CH
3
)
2
). C-NMR (125 MHz, CD
3
OD): δ 156.4, 154.7, 152.5, 140.1, 120.8,
8
9.8, 86.8, 73.0, 63.7, 57.5, 45.8, 41.6, 37.6, (one broadened resonance could not be identified). HRMS
+
(
TOF) calcd for C15
H24ClN
6
3
O [M + H] 371.1598, found 371.1584.
3
.5. General Procedure for the Desilylation of Ribose Cladribine Analogues
To a 0.1 M solution of the silylated compound in anhydrous MeOH, KF (2 equiv/silyl group) was
added. The mixture was heated at 80 °C for 24 h, cooled, and silica gel was added. The mixture was
evaporated to dryness and the compound-impregnated silica gel was loaded onto a wet-packed silica gel
column. The products were obtained by elution with appropriate solvents (see the individual compound
headings for details).
2
-Chloroadenosine (12b): Prepared from compound 5b (100 mg, 0.155 mmol) and KF (54.0 mg,
0
.93 mmol) in MeOH (1.55 mL). Chromatography on a silica gel column sequentially eluted with 5%
MeOH in EtOAc and 10% MeOH in EtOAc gave 35.0 mg (74% yield) of compound 12b as an
1
off-white solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.13. H-NMR (400 MHz, CD
3
OD): δ 8.27 (s,
1
H, H-8), 5.92 (d, J = 6.0 Hz, 1H, H-1′), 4.72 (t, J = 5.6, 1H, H-2′), 4.32 (dd, J = 2.8, 5.2 Hz, 1H,
H-3′), 4.14 (m, 1H, H-4′), 3.90 (dd, J = 2.8, 12.4 Hz, 1H, H-5′), 3.76 (dd, J = 2.8, 12.4 Hz, 1H, H-5′).
13C-NMR (100 MHz, DMSO-d
): δ 174.5, 156.7, 152.9, 150.3, 140.0, 118.1, 87.4, 85.7, 73.8, 70.3, 61.3,
6
+
2
5.3. HRMS (TOF) calcd for C10
H13ClN
5
O
4
[M + H] 302.0651, found 302.0627.
6
2
-Chloro-N -methyladenosine (13b): Prepared from compound 6b (100 mg, 0.152 mmol) and KF
(52.9 mg, 0.912 mmol) in MeOH (1.52 mL). Chromatography on a silica gel column sequentially eluted
with 2.5% MeOH in EtOAc and 5% MeOH in EtOAc gave 36.0 mg (75% yield) of compound 13b as a
1
white, foamy solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.21. H-NMR (400 MHz, CD
3
OD): δ 8.20
(s, 1H, H-8), 5.90 (d, J = 6.0 Hz, 1H, H-1′), 4.68 (t, J = 5.6 Hz, 1H, H-2′), 4.31 (dd,
J = 2,8, 4.8 Hz, 1H, H-3′), 4.14 (m, 1H, H-4′), 3.96 (dd, J = 2.4, 12.4 Hz, 1H, H-5′), 3.76 (dd, J = 2.8, 12.4
1
3
Hz, 1H, H-5′), 3.07 (br s, 3H, NCH
3
). C-NMR (100 MHz, DMSO-d ): δ 155.5, 153.2, 149.2, 139.7,
6
1
18.6, 87.3, 85.6, 73.6, 70.6, 70.3, 61.6, 61.3, 53.9, 27.1. HRMS (TOF) calcd for C11
H15ClN
5
O
4
+
[M + H] 316.0807, found 316.0808.
6
6
2
-Chloro-N ,N -dimethyladenosine (14b): Prepared from compound 7b (100 mg, 0.148 mmol) and KF
(
51.8 mg, 0.892 mmol) in MeOH (1.48 mL). Chromatography on a silica gel column sequentially eluted
with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave 36.8 mg (75% yield) of compound
1
1
4b as an off-white, foamy solid. R
f
(SiO
2
and 5% MeOH in EtOAc) = 0.32. H-NMR
(400 MHz, CD
3
OD): δ 8.17 (s, 1H, H-8), 5.91 (d, J = 6.4 Hz, 1H, H-1′), 4.67 (t, J = 5.4 Hz, 1H, H-2′),
4
.31 (dd, J = 2.8, 4.8 Hz, 1H, H-3′), 4.14 (m, 1H, H-4′), 3.90 (dd, J = 2.8, 12.4 Hz, 1H, H-5′), 3.76 (dd,
1
3
J = 2.4, 12.4 Hz, 1H, H-5′), 3.60–3.49 (br m, H, N(CH
3
)
2
). C-NMR (100 MHz, DMSO-d ): δ 154.4,
6
1
52.5, 151.1, 138.6, 118.5, 87.2, 85.6, 73.6, 70.2, 61.2, 37.5 (br). HRMS (TOF) calcd for C12
H17ClN
5
O
4
+
[M + H] 330.0964, found 330.0964.

Molecules 2015, 20
18454
2-Chloro-6-(pyrrolidin-1-yl)-9-(β-D-ribofuranosyl)purine (15b): Prepared from compound 8b (100 mg,
0.143 mmol) and KF (49.8 mg, 0.858 mmol) in MeOH (1.43 mL). Chromatography on a silica gel
column sequentially eluted with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave 43.3 mg
1
(
85% yield) of compound 15b as a white solid. R
f
(SiO
2
and 5% MeOH in EtOAc) = 0.38. H-NMR (400
3
MHz, CD OD): δ 8.18 (s, 1H, H-8), 5.91 (d, J = 6.4 Hz, 1H, H-1′), 4.67 (t, J = 5.4 Hz, 1H, H-2′), 4.32 (dd,
J = 2.8, 4.8 Hz, 1H, H-3′), 4.14 (m, 1H, H-4′), 4.13–4.10 (br m, 2H, pyrrolidinyl NCH), 3.90 (dd,
J = 2.8, 12.8 Hz, 1H, H-5′), 3.74 (dd, J = 2.4, 12.4 Hz, 1H, H-5′), 3.72–3.65 (br m, 2H, pyrrolidinyl
NCH), 2.13–2.07 (br m, 2H, pyrrolidinyl CH), 2.05–1.95 (br m, 2H, pyrrolidinyl CH). 13C-NMR
(
100 MHz, DMSO-d
6
): δ 152.7, 152.7, 150.7, 139.1, 118.7, 87.2, 85.6, 73.7, 70.2, 61.2, 48.6, 47.3, 25.6,
+
2
3.6. HRMS (TOF) calcd for C14
H19ClN
5
O
4
[M + H] 356.1120, found 356.1127.
2
-Chloro-6-(piperidin-1-yl)-9-(β-D-ribofuranosyl)purine (16b): Prepared from compound 9b (100 mg,
0.140 mmol) and KF (48.9 mg, 0.842 mmol) in MeOH (1.40 mL). Chromatography on a silica gel
column sequentially eluted with EtOAc and 2% MeOH in EtOAc gave 43.6 mg (84% yield) of
1
compound 16b as a white, foamy solid. R
400 MHz, CD
.27 (dd, J = 2.8, 5.2 Hz, 1H, H-3′), 4.19 (br s, 4H, piperidinyl N(CH
J = 2.8, 12.2 Hz, 1H, H-5′), 3.73 (dd, J = 2.4, 12.2 Hz, 1H, H-5′), 1.74 (br m, 2H, piperidinyl CH
f
(SiO
2
and 10% MeOH in EtOAc) = 0.41. H-NMR
(
3
OD): δ 8.13 (s, 1H, H-8), 5.87 (d, J = 6.0 Hz, 1H, H-1′), 4.64 (t, J = 5.4 Hz, 1H, H-2′),
), 4.11 (m, 1H, H-4′), 3.89 (dd,
), 1.64
): δ 153.1, 152.6, 151.4, 138.5, 118.2, 87.2,
5.6, 73.6, 70.4, 70.2, 70.1, 61.2, 44.8 (br), 25.6, 25.2, 23.9, 14.0. HRMS (TOF) calcd for C15
4
)
2 2
2
1
3
(
br m, 4H, piperidinyl CH
2
). C-NMR (100 MHz, DMSO-d
6
8
H21ClN
5
O
4
+
[M + H] 370.1277, found 370.1302.
2-Chloro-6-(morpholin-4-yl)-9-(β-D-ribofuranosyl)purine (17b): Prepared from compound 10b (100 mg,
0.139 mmol) and KF (48.7 mg, 0.839 mmol) in MeOH (1.40 mL). Chromatography on a silica gel column
sequentially eluted with 50% EtOAc in hexanes, EtOAc, and 10% MeOH in EtOAc gave 40.6 mg (78%
1
yield) of compound 17b as a white, foamy solid. R
f
(SiO
2
and 5% MeOH in EtOAc) = 0.21. H-NMR
(
400 MHz, CD
3
OD): δ 8.21 (s, 1H, H-8), 5.92 (d, J = 6.0 Hz, 1H, H-1′), 4.66 (t, J = 5.6 Hz, 1H, H-2′),
.32 (dd, J = 2.8, 4.8 Hz, 1H, H-3′), 4.30–4.24 (br m, 4H, morpholinyl N(CH ), 4.14 (m, 1H, H-4′),
.90 (dd, J = 2.8, 12.4 Hz, 1H, H-5′), 3.80 (t, J = 4.8 Hz, 4H, morpholinyl CH OCH ), 3.76 (dd, J = 2.8,
): δ 153.3, 152.5, 151.6, 139.0, 118.3, 87.3, 85.6,
4
3
1
8
3
)
2 2
2
2
1
3
2.4 Hz, 1H, H-5′). C-NMR (100 MHz, DMSO-d
6
5.4, 73.7, 70.4, 70.2, 66.0, 61.4, 61.1, 48.5, 45.0 (br). HRMS (TOF) calcd for C14
72.1069, found 372.1056.
H19ClN
5
O
[M + H]+
5
2-Chloro-6-[(2-dimethylamino)ethyl)(methyl)amino)]-9-(β-D-ribofuranosyl)purine (18b): Prepared from
compound 11b (100 mg, 0.137 mmol) and KF (47.7 mg, 0.822 mmol) in MeOH (1.37 mL).
Chromatography on a silica gel column sequentially eluted with 10% MeOH in EtOAc and 20% MeOH
in EtOAc gave 34.5 mg (65% yield) of compound 18b as a white, foamy solid. R
f
(SiO
OD): δ 8.19 (s, 1H, H-8), 5.92 (d, J = 6 Hz, 1H, H-1′), 4.67 (t,
H, J = 5.6 Hz, 1H, H-2′), 4.34 (dd, J = 3.2, 5.2 Hz 1H, H-3′), 4.14 (d, J = 2.4 Hz, 3H, H-4′), 3.90 (dd,
J = 2.4, 12.4 Hz, 1H, H-5′), 3.76 (dd, J = 2.8, 12.4 Hz, 1H, H-5′), 3.49 (br s, 3H, NCH ), 2.83 (t, 2H,
OD): δ 156.1, 154.7, 152.3, 140.5,
2
and 15% MeOH in
1
EtOAc) = 0.10. H-NMR (400 MHz, CD
3
1
3
1
3
NCH2, J = 6.8 Hz ), 2.47 (s, 6H, N(CH
3
)
2
). C-NMR (100 MHz, CD
3

Molecules 2015, 20
18455
1
20.7, 90.8, 87.7, 75.3, 72.3, 63.2, 57.0, 49.6, 49.4, 49.2, 49.0, 48.7, 48.5, 48.3, 45.5, 37.4. HRMS (TOF)
+
calcd for C15
H24ClN
6
4
O [M + H] 387.1542, found 387.1513.
6
O -(Benzotriazol-1-yl)-2-bromo-9-[2-deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine
(
19a): A mixture of compound 3a (300.0 mg, 0.489 mmol) and SbBr
CH Br (4.9 mL) was cooled to −15 °C using dry ice and acetone, in a nitrogen atmosphere. t-BuONO
203.8 μL, 1.713 mmol) was added dropwise and the mixture was stirred at −10 °C to −15 °C for 2 h.
Because TLC indicated the presence of starting material, another aliquot of t-BuONO (203.8 μL,
.713 mmol) was added and the reaction proceeded for 1 h at −15 °C, at which time TLC indicated the
reaction to be complete. The reaction mixture was poured into ice-cold, saturated aqueous NaHCO
5 mL) with stirring. The mixture was filtered using vacuum (note: use of vacuum for this filtration is
critical for maximizing product recovery) and the residue was washed with CH Cl (5 mL). The organic
layer was separated and the aqueous layer was back extracted with CH Cl (2 × 5 mL). The combined
organic layer was washed with water (5 mL) and brine (5 mL), dried over anhydrous Na SO , and
3
(247.7 mg, 0.685 mmol) in dry
2
2
(
1
3
(
2
2
2
2
2
4
evaporated under reduced pressure. Purification of the crude material on a silica gel column sequentially
eluted with hexanes, 5% EtOAc in hexanes, and 30% EtOAc in hexanes gave 208.5 mg (63% yield) of
1
compound 19a as a white foam. R
f
(SiO
2
and 30% EtOAc in hexanes) = 0.60. H-NMR (500 MHz,
3
CDCl ): δ 8.45 (s, 1H, H-8), 8.13 (d, J = 8.3 Hz, 1H, Ar-H), 7.56–7.45 (m, 3H, Ar-H), 6.47 (t, J = 5.9 Hz,
1
1
0
1
H, H-1′), 4.63 (m, 1H, H-3′), 4.03 (m, 1H, H-4′), 3.90 (dd, J = 3.4, 11.2 Hz, 1H, H-5′), 3.78 (dd, J = 1.5,
1.2 Hz, 1H, H-5′), 2.61 (app quint, Japp ~ 6.2 Hz, 1H, H-2′), 2.49 (app quint, Japp ~ 5.8 Hz, 1H, H-2′),
13
.91 (s, 18H, t-Bu), 0.11 and 0.09 (2s, 12H, SiCH
3
). C-NMR (125 MHz, CDCl ): δ 158.7, 154.9, 144.3,
3
43.6, 142.6, 129.1, 128.9, 125.1, 120.8, 119.5, 108.7, 88.5, 85.4, 71.7, 62.7, 41.8, 26.2, 25.9, 18.6, 18.2,
+
−
4.4, −4.6, −5.2, −5.3. HRMS (TOF) calcd for C28H43BrN O Si
7 4 2
[M + H] 676.2093, found 676.2078.
2
-Bromo-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyadenosine (20a): To a solution of compound 19a
(
135.3 mg, 0.20 mmol) in 1,2-DME (2 mL), 28%–30% aqueous ammonia (48.6 μL) was added, and the
mixture was stirred at room temperature for 45 min. The mixture was diluted with EtOAc (15 mL) and
washed with 5% aqueous NaCl (10 mL). The organic layer was separated and the aqueous layer was
back extracted with EtOAc (15 mL). The combined organic layer was dried over anhydrous Na
2
SO and
4
evaporated under reduced pressure. The crude material was chromatographed on a silica gel column by
sequential elution with hexanes, 20% EtOAc in hexanes and 40% EtOAc in hexanes to afford 93.1 mg
1
(
83% yield) of compound 20a as a white foam. R
δ 8.08 (s, 1H, H-8), 6.63 (br s, 2H, Ar-NH
H, H-4′), 3.88 (dd, J = 3.9, 11.2 Hz, 1H, H-5′), 3.75 (dd, J = 2.4, 11.2 Hz, 1H, H-5′), 2.61 (app quint,
app ~ 6.3 Hz, 1H, H-2′), 2.43–2.38 (m, 1H, H-2′), 0.90 (s, 18H, t-Bu), 0.09 and 0.08 (2s, 12H, SiCH ).
13C-NMR (125 MHz, CDCl
): δ 156.3, 150.4, 144.9, 139.6, 119.3, 88.2, 84.7, 71.9, 62.9, 41.4, 26.1, 25.9,
8.6, 18.2, −4.4, −4.6, −5.2, −5.3. HRMS (TOF) calcd for C22
found 558.1902.
f
(SiO
2
and EtOAc) = 0.50. H-NMR (500 MHz, CDCl
3
):
2
), 6.37 (t, J = 6.1 Hz, 1H, H-1′), 4.61 (m, 1H, H-3′), 3.98 (m,
1
J
3
3
+
1
H41BrN
5
O
3
Si [M + H] 558.1926,
2
2
-Bromo-2′-deoxyadenosine (21a): As described in the general desilylation procedures, compound 21a
was prepared from compound 20a (80.0 mg, 0.143 mmol) and KF (33.2 mg, 0.572 mmol) in MeOH
1.4 mL), over 28 h. Chromatography on a silica gel column sequentially eluted with 50% EtOAc in
(

Molecules 2015, 20
18456
hexanes, EtOAc, and 10% MeOH in EtOAc gave 31.1 mg (66% yield) of compound 21a as a white/
1
off-white solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.40. H-NMR (500 MHz, CD
3
OD): δ 8.25 (s, 1H,
H-8), 6.36 (t, J = 6.8 Hz, 1H, H-1′), 4.57 (m, 1H, H-3′), 4.04 (m, 1H, H-4′), 3.84 (dd, J = 2.9, 12.2 Hz, 1H,
H-5′), 3.74 (dd, J = 3.4, 12.2 Hz, 1H, H-5′), 2.76 (app quint, Japp ~ 6.7 Hz, 1H, H-2′), 2.43–2.39 (ddd,
1
3
J = 2.9, 5.9, 13.2 Hz, 1H, H-2′). C-NMR (125 MHz, CD
3
OD): δ 158.1, 151.4, 145.9, 141.7, 120.3,
+
8
9.9, 86.9, 72.9, 63.7, 41.6. HRMS (TOF) calcd for C10
H12BrN
5
O
3
Na [M + Na] 352.0016,
found 352.0021.
6
O -(Benzotriazol-1-yl)-2-bromo-9-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (19b):
A mixture of compound 3b (500.0 mg, 0.67 mmol) and SbBr
3
(340.8 mg, 0.94 mmol) in dry CH
8.3 mL) was cooled to −15 °C using dry ice and acetone, in a nitrogen atmosphere. t-BuONO
280 μL, 2.352 mmol) was added dropwise and the mixture was stirred at −10 °C to −15 °C for 2 h.
2
Br
2
(
(
Because TLC indicated the presence of starting material, another aliquot of t-BuONO (280 μL,
.352 mmol) was added and the reaction was allowed to progress for 1 h at −15 °C, at which time TLC
indicated the reaction to be complete. The reaction mixture was poured into ice-cold, saturated aqueous
NaHCO (10 mL) with stirring. The mixture was filtered using vacuum (note: use of vacuum for this
filtration is critical for maximizing product recovery) and the residue was washed with CH Cl (15 mL).
The organic layer was separated and the aqueous layer was back extracted with CH Cl
2 × 15 mL). The combined organic layer was washed with water (10 mL) and brine (10 mL), dried over
anhydrous Na SO , and evaporated under reduced pressure. Purification of the crude material on a silica
gel column sequentially eluted with hexanes, 5% EtOAc in hexanes, and 30% EtOAc in hexanes gave
50 mg (64% yield) of compound 19b as a white foam. R (SiO and 30% EtOAc in hexanes) = 0.80.
1H-NMR (400 MHz, CDCl
): δ 8.54 (s, 1H, H-8), 8.15 (d, J = 8.4 Hz, 1H, Ar-H), 7.58–7.45 (m, 3H,
Ar-H), 6.05 (d, J = 4.0 Hz, 1H, H-1′), 4.57 (t, J = 4.2 Hz, 1H, H-2′), 4.33 (t, J = 4.6 Hz, 1H, H-3′), 4.17 (m,
2
3
2
2
2
2
(
2
4
3
f
2
3
1
0
1
2
H, H-4′), 4.08 (dd, J = 3.6, 11.6 Hz, 1H, H-5′), 3.83 (dd, J = 2.4, 11.6 Hz, 1H, H-5′), 0.96, 0.93, and
1
3
.85 (3s, 27H, t-Bu), 0.16, 0.11, −0.03, and −0.09 (4s, 18H, SiCH
3
). C-NMR (100 MHz, CDCl ): δ
3
58.5, 154.8, 144.2, 143.4, 142.5, 128.8, 128.7, 124.8, 120.6, 119.4, 108.5, 89.5, 85.2, 76.1, 71.1, 61.9,
9.6, 26.1, 25.8, 25.6, 18.5, 18.0, 17.9, −4.2, −4.6, −4.7, −4.9, −5.3, −5.4. HRMS (TOF) calcd for
+
C
34
H57BrN
7
O
5
Si
3
[M + H] 806.2907, found 806.2912.
2
-Bromo-2′,3′,5′-tri-O-(t-butyldimethylsilyl)-adenosine (20b): To a solution of compound 19b
(
200 mg, 0.247 mmol) in 1,2-DME (4 mL), 28%–30% aqueous ammonia (60 μL) was added, and the
mixture was stirred at room temperature for 45 min. The mixture was diluted with EtOAc (20 mL) and
washed with 5% aqueous NaCl (20 mL). The organic layer was separated and the aqueous layer was
back extracted with EtOAc (25 mL). The combined organic layer was dried over anhydrous Na
2
SO and
4
evaporated under reduced pressure. The crude material was chromatographed on a silica gel column by
sequential elution with hexanes, 20% EtOAc in hexanes, and 40% EtOAc in hexanes to afford 140 mg
1
(
82% yield) of compound 20b as a white foam. R
CDCl ): δ 8.10 (s, 1H, H-8), 5.92 (d, J = 4.8 Hz, 1H, H-1′), 5.77 (br s, 2H, Ar-NH
H, H-2′), 4.32 (t, J = 4.2 Hz, 1H, H-3′), 4.13 (m, 1H, H-4′), 4.06 (dd, J = 4.8, 11.2 Hz, 1H, H-5′), 3.80
dd, J = 2.8, 11.2 Hz, 1H, H-5′), 0.95, 0.93, and 0.83 (3s, 27H, t-Bu), 0.14, 0.11, −0.00, and −0.15 (4s,
f
(SiO
2
and EtOAc) = 0.55. H-NMR (400 MHz,
3
2
), 4.69 (t, J = 4.6 Hz,
1
(
1
3
1
8H, SiCH
3
). C-NMR (100 MHz, CDCl ): δ 155.8, 150.5, 144.6, 140.0, 119.4, 110.0, 89.0, 85.3, 75.4,
3

Molecules 2015, 20
18457
7
1.7, 62.3, 26.0, 25.8, 25.7, 18.4, 18.0, 17.9, −4.3, −4.7, −5.0, −5.3, −5.4. HRMS (TOF) calcd for
+
C
28
H55BrN
5
O
4
Si [M + H] 688.2740, found 688.2728.
3
2
-Bromoadenosine (21b): As described in the general desilylation procedures, compound 21b was
prepared from compound 20b (100 mg, 0.145 mmol) and KF (50.5 mg, 0.870 mmol) in MeOH
1.9 mL), over 24 h. Chromatography on a silica gel column sequentially eluted with 50% EtOAc in
(
hexanes, EtOAc, and 10% MeOH in EtOAc which gave 34.9 mg (69% yield) of compound 21b as a pale
1
yellow solid. R
f
(SiO
2
and 10% MeOH in EtOAc) = 0.39. H-NMR (400 MHz, CD
3
OD): δ 8.25 (s, 1H,
H-8), 5.92 (d, J = 6.4 Hz, 1H, H-1′), 4.67 (d, J = 5.6 Hz, 1H, H-2′), 4.32 (dd, J = 2.8, 4.8 Hz, 1H, H-3′),
.15 (m, 1H, H-4′), 3.90 (dd, J = 2.8, 12.4 Hz, 1H, H-5′), 3.76 (dd, J = 2.8, 12.4 Hz, 1H,
4
13
H-5′). C-NMR (100 MHz, CD
3
OD): δ 156.5, 150.2, 144.1, 139.8, 118.4, 87.2, 85.7, 73.6, 70.3, 61.3.
+
HRMS (TOF) calcd for C10
H
13BrN
5
O
4
[M + H] 346.0145, found 346.0154.
3
.6. Protocols for Tests against HCL, TCL, and CCL
Blood was obtained in sodium heparin tubes from patients as part of protocols with consent forms
approved by the investigators review board (IRB) of the National Cancer Institute. The blood was diluted
:1 with PBS without calcium or magnesium, layered over 15 mL Ficoll in 50 mL tubes, and centrifuged
1
to obtain mononuclear cells. Patients with high lymphocytosis had leukemic cells >80%–90% pure after
Ficoll. The cells were viably frozen in 7.5% DMSO in leucine-poor media (LPM, 88% leucine-free RPMI,
2
% RPMI, and 10% FBS) in cryovials and stored under liquid nitrogen. LPM also contained penicillin,
streptomycin, glutamine, gentamycin, and doxycycline. To assay, thawed cells were washed, suspended
in LPM, added to 96-well round-bottom plates (15 μL/well), and treated with 15 μL of purine analogues
diluted in LPM. The aliquots were incubated 3 days, then treated with 10 μL of either ATP (CellTiter-Glo,
3
Promega, Madison, WI, USA) or { H}-leucine (Perkin-Elmer, Waltham, MA, USA) diluted in leucine-free
3
RPMI. After 30 min of ATP, the plate is read for bioluminescence. After 6 h of { H}-leucine, the cells
were liberated by freeze-thaw, harvested on to glass-fiber filters, counted either by a beta scintillation
counter. The number of cells cultured in 30 μL aliquots for ATP assay was 20,000, 20,000, and 100,000
3
for HCL, TCL, and CLL, respectively. The cell number for { H}-leucine assay was 60,000, 60,000 and
3
2
00,000 for HCL, TCL, and CLL, respectively. HCL and TCL cells were pulsed with 1 μCi of { H}-leucine,
while CLL cells were pulsed with 1.5–2 μCi/well. The IC50 was the calculated concentration needed for
3
5
0% inhibition, defined as the ATP uptake or { H}-leucine incorporation corresponding to halfway
between that of control (cells with LPM alone) and that of cycloheximide 10 μg/mL. Reported IC50
values were the means of 3 triplicate experiments.
3
.7. Protocols for Tests against Breast Cancer Cells
MDA-MB-231 breast cancer (BC) cells were obtained from the American Type Culture Collection
(
ATCC, Manassas, VA, USA), and were cultured at 37 °C in 5% CO using culture medium recommended
2
by the supplier. Each tested compound was dissolved in sterile DMSO, then diluted (4.8–57.7 mM) in
5
sterile 1X PBS for a final DMSO concentration of 0.1%. Then, 2 × 10 cells/well, were seeded and cultured
for 24 h as described [51,52]. Afterwards, the media was changed to 5% FBS for 1 h and cells were treated
in duplicate with dilutions of each treatment (0–1.8 mM) for 24 h. Cells were fixed (cold methanol), and

Molecules 2015, 20
18458
nuclei stained (0.4% propidium iodide, (PI)) (Sigma-Aldrich, St Louis, MO, USA), and measured using
®
a GloMax Microplate Reader (Promega, Madison, WI, USA). Cell viability was calculated as percent
of surviving cells after treatment relative to vehicle wells. The IC50 was obtained from dose response
®
curve fittings using the non-linear regression function of GraphPad Prism version 6.0b for Mac
(
GraphPad Software, San Diego, CA, USA) [53].
. Conclusions
We have demonstrated, for the first time, the synthesis of O -(benzotriazol-1-yl)-2-chloro-9-[2-
4
6
deoxy-3,5-di-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]purine (4a) by diazotization-chlorination of
6
O -(benzotriazol-1-yl)-3′,5′-di-O-(t-butyldimethylsilyl)-2′-deoxyguanosine (3a) with t-BuONO and SbCl
3
in CH
2
Cl
2
. This procedure afforded better yields than the chlorination using t-BuONO and
6
Me SiCl. This compound and its ribose analogue, O -(benzotriazol-1-yl)-2-chloro-9-[2,3,5-tri-O-(t-
3
butyldimethylsilyl)-β-D-ribofuranosyl]purine, both undergo smooth reactions with ammonia, and
primary, and secondary amines to produce cladribine (12a), its N-modified analogues (13a–18a), and the
corresponding ribose derivatives (12b–18b), after a simple desilylation with KF in MeOH. These
compounds were tested against HCL, TCL, and CLL, but none of the new compounds was more active
than cladribine itself. The bromo as well as ribose analogues of cladribine displayed activity but the
bromo analogue of cladribine was more active against TCL and CLL as compared to both the ribose
equivalent and the bromo ribose analogue of cladribine. The compound containing both the bromine
atom and a ribose ring was least active among the compounds possessing a primary amino group at the
C6 position. Thus, it appears that a free amino group at this location is critical to the activity of cladribine.
Interestingly, the C6 piperidinyl analog of cladribine showed low activity. Tests against MDA-MB-231
breast cancer cells showed that only cladribine and its ribose analogue showed some activity. The bromo
analogues were about 10 times less active and all others showed no potential. Despite the lack of a major
improvement in the activity of cladribine, or the identification of new compounds with activity against
breast cancer, this work has provided a route to four doubly functionalizable nucleoside derivatives. The
orthogonal reactivities of these compounds, i.e., S Ar at the C6 position and metal catalysis at the C2
N
position, can be used for development of novel nucleoside analogues. We anticipate pursuing further
work along these lines in the future.
Supplementary Materials
Supplementary can be accessed at: http://www.mdpi.com/1420-3049/20/10/18437/s1.
Acknowledgments
This work was supported by National Science Foundation Grant CHE-1265687 to MKL, in part by
National Institutes of Health Grant SC3GM111171 to MMMM from the National Institute on General
Medical Sciences, and Title V PPOHA US Department of Education P031M105050 to Universidad
Central del Caribe. Infrastructural support at CCNY was provided by National Institute on Minority Health
and Health Disparities Grant G12MD007603, while that at UCC was provided by G12MD007583. Siva
Subrahmanyam Relangi, Venkateshwarlu Gurram, Muralidharan Kaliyaperumal, Somesh Sharma, and

Molecules 2015, 20
18459
Narender Pottabathini gratefully acknowledge support from GVKBIO and Siva Subrahmanyam Relangi
thanks Anna Venkateswara Rao (Koneru Lakshmaiah University, Guntur (Andhra Pradesh), India) for
his support.
Author Contributions
Sakilam Satishkumar, Prasanna K. Vuram, and Siva Subrahmanyam Relangi had equal contributions to
the synthesis portion of this work. Sakilam Satishkumar and Prasanna K. Vuram (City College of New York)
performed optimization of the synthetic procedures, executed syntheses of the deoxyribose series,
performed spectroscopic analyses of the compounds, and produced a part of the experimental section.
Messrs. Relangi and Venkateshwarlu Gurram (GVKBIO) executed syntheses of the ribose series,
performed spectroscopic analyses of the compounds, and produced a part of the experimental section.
Hong Zhou and Robert J. Kreitman (National Cancer Institute) tested the compounds against HCL, TCL,
and CLL. Michelle M. Martínez Montemayor tested the compounds against breast cancer cell lines. Lijia
Yang (City College of New York) performed HRMS analysis of all compounds synthesized in
Lakshman’s laboratories. Narender Pottabathini (GVKBIO) was responsible for the oversight of the
work performed in his laboratories. Mahesh K. Lakshman (City College of New York) conceived and
designed the research, assisted with data analysis, and wrote a significant portion of this manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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3. GraphPad Software. Available online: http://www.graphpad.com.
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Sample Availability: Limited amounts of compounds 12a–18a and 12b–18b may be available from
the authors.
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2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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