Design, Synthesis, and Activity Evaluation of Novel Acyclic Nucleosides as Potential Anticancer Agents in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.0c01717

In the present work, 103 novel acyclic nucleosides were designed, synthesized, and evaluated for their anticancer activities in vitro and in vivo. The structure-activity relationship (SAR) studies revealed that most target compounds inhibited the growth of colon cancer cells in vitro, of which 3-(6-chloro-9H-purin-9-yl)dodecan-1-ol (9b) exhibited the most potent effect against the HCT-116 and SW480 cells with IC50 values of 0.89 and 1.15 μM, respectively. Furthermore, all of the (R)-configured acyclic nucleoside derivatives displayed more potent anticancer activity compared to their (S)-counterparts. Mechanistic studies revealed that compound 9b triggered apoptosis in the cancer cell lines via depolarization of the mitochondrial membrane and effectively inhibited colony formation. Importantly, compound 9b inhibited the growth of the SW480 xenograft in a mouse model with low systemic toxicity. These results indicated that acyclic nucleoside compounds are viable as potent and effective anticancer agents, and compound 9b may serve as a promising lead compound that merits further attention in future anticancer drug discovery.

Full text of DOI:10.1021/acs.jmedchem.0c01717

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Design, Synthesis, and Activity Evaluation of Novel Acyclic
Nucleosides as Potential Anticancer Agents In Vitro and In Vivo
§
§
§
Er-Jun Hao, Gong-Xin Li, Yu-Ru Liang, Ming-Sheng Xie, Dong-Chao Wang, Xiao-Han Jiang,
Jia-Yi Cheng, Zhi-Xian Shi, Yang Wang,* and Hai-Ming Guo*
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sı Supporting Information
ABSTRACT: In the present work, 103 novel acyclic nucleosides were designed, synthesized, and evaluated for their anticancer
activities in vitro and in vivo. The structure−activity relationship (SAR) studies revealed that most target compounds inhibited the
growth of colon cancer cells in vitro, of which 3-(6-chloro-9H-purin-9-yl)dodecan-1-ol (9b) exhibited the most potent effect against
the HCT-116 and SW480 cells with IC50 values of 0.89 and 1.15 μM, respectively. Furthermore, all of the (R)-configured acyclic
nucleoside derivatives displayed more potent anticancer activity compared to their (S)-counterparts. Mechanistic studies revealed
that compound 9b triggered apoptosis in the cancer cell lines via depolarization of the mitochondrial membrane and effectively
inhibited colony formation. Importantly, compound 9b inhibited the growth of the SW480 xenograft in a mouse model with low
systemic toxicity. These results indicated that acyclic nucleoside compounds are viable as potent and effective anticancer agents, and
compound 9b may serve as a promising lead compound that merits further attention in future anticancer drug discovery.
1
. INTRODUCTION
acyclic nucleosides containing 2,3-epoxypropyl, 3-amino-2-
hydroxypropyl, or 2,3-epoxypropyl ether moieties and their
́ ́
antitumor and antiviral activities. Later, Raic-Malic et al.
Acyclic nucleoside compounds (ANCs) are one of the most
versatile scaffolds in medicinal chemistry owing to their unique
physicochemical and biological properties. The absence of a
glycosidic bond increases the resistance of ANCs to chemical
and biological degradation,
allows structural adaptability for interacting with the active site
22
reported the synthesis and biological evaluation of fluorinated
and iodinated acyclic purine nucleoside analogues bearing 9-
(2-hydroxypropyl) and 9-(2-hydroxyethoxymethyl) side
1
−6
and the flexible acyclic chain
23
chains. In 2006, Mintas et al. reported the synthesis of
unsaturated acyclic and epoxide nucleoside analogues and
7
−15
of multiple enzymes.
Over the last few decades, R&D in
24,25
evaluation of their antiviral and cytostatic activities.
015, Raic-Malic et al. reported the discovery of new acid
ceramidase-targeted acyclic 5-alkynyl and 5-heteroaryl uracil
In
ANCs has resulted in the successful launch of several approved
antiviral drugs (e.g., Ganciclovir, Famciclovir, Penciclovir,
Figure 1) and is still providing a number of novel bioactive
compounds.
2
́
́
2
6
nucleosides. Although some studies on the antitumor
27−33
activities of acyclic nucleosides have been reported,
in
The mature acyclic nucleoside preparation methods have
16,17
view of the frequent occurrence of chemoresistance against the
current anticancer drugs, there is an urgent demand for
developing novel chemotherapeutic agents with high potency
and low toxicity. Based on our extensive research work on the
provided a convenient synthesis of ANCs,
been proved to possess excellent bioactivities. Since the
pioneer work of De Clercq and Holy in 1976 at the symposium
on synthetic nucleosides, nucleotides, and polynucleotides,
acyclic nucleoside phosphonates have been recognized as a key
class of antiviral agents. In 1977, Acyclovir with an acyclic
side chain, 2-hydroxyethoxymethyl at the N9 position, was first
discovered as an antiherpetic agent.
and Holy reported an acyclic nucleoside, (S)-9-(2,3-
dihydroxypropyl)adenine, with broad-spectrum antiviral activ-
which have
́
18
Received: September 30, 2020
Published: February 4, 2021
19,20
In 1978, De Clercq
́
2
1
ity. In 1999, Raic-Malic et al. developed the synthesis of
́ ́
©
2021 American Chemical Society
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Figure 1. Acyclic nucleoside and nucleotide drugs.
Figure 2. Design of acyclic nucleosides.
synthetic methodology of acyclic nucleosides, 4−36 we will
study the antitumor activities of the synthesized acyclic
nucleosides. To this end, we constructed a library of 103
novel acyclic nucleoside derivatives and evaluated their
anticancer activities through established in vitro and in vivo
assays.
3
the C1′-position, which form a chiral center, or a CC double
bond at the C2′-position (Figure 2b). Finally, the C2, C6, and
C8 positions of the purine ring could also be altered. It should
be noted that the above studies on the structure−activity
relationship (SAR) of ANCs are all aimed at antiviral activities,
and the SAR of antitumor activities of ANCs is less reported.
In particular, the SAR of our designed ANCs containing a side
chain with a length of three carbon atoms has not been
systematically reported. To probe into the SAR of our designed
acyclic purine nucleoside analogues, a molecular library of 103
acyclic nucleosides was synthesized and the anticancer
activities were evaluated.
2
. DESIGN
First, the side chains of Penciclovir and Adefovir are
structurally analyzed. In addition to a hydroxyl group, the
length of the side chain is four and two carbon atoms,
respectively (Figure 2a). Therefore, we design the acyclic
nucleosides containing a side chain with a length of three
carbon atoms and a hydroxyl group (Figure 2b). Second, when
a methyl group is introduced into the C2′-position of Adefovir,
a chiral carbon emerges and a new acyclic nucleotide drug
Tenofovir is formed (Figure 2a). Thus, we design the acyclic
nucleosides bearing a side chain with different alkyl groups at
3. CHEMISTRY
At first, all compounds were synthesized and isolated as
racemic mixtures. Except for 1b, 3b, 16b, 4c, 5c, 6c, 5k, and
10k, all ANCs generated in the compound library were novel.
Acyclic nucleoside compounds 1a−12a, 1b−15b, 4c−9c, 4d−
2
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Scheme 1. Syntheses of 1a−13a, 1b−16b, 4c−9c, 4d−11d, 5e−11e, 5f−11f, 4g−11g, 5h−11h, 5i−9i, 4j−6j, and 5k−10k
2
079
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Scheme 1. continued
Scheme 2. Asymmetric Syntheses of (R)- and (S)-configurations of the compounds 7b, 8b, and 9b
1
1d, 5e−11e, 5f−11f, and 5i−9i were synthesized in 26.7−
organocatalyst (Cat. II) and subsequent reduction with sodium
borohydride (Scheme 2). The S enantiomers were obtained
using catalyst III with opposite configuration at 50.5−54.8%
overall yield and 91−95% ee (Scheme 2). In addition, the
absolute configuration of the compound (R)-9b was
determined via single-crystal X-ray diffraction analysis (SI,
Figure S6).
8
9.5% overall yields via L-proline (Cat. I)-catalyzed aza-
Michael addition of purine bases to α,β-unsaturated aldehydes,
followed by treatment of the resulting Michael adducts with
NaBH in MeOH at 0 °C (Scheme 1). In addition, the
structure of the compounds 6a and 8b was determined via
single-crystal X-ray diffraction analysis (Supporting Informa-
tion (SI), Figures S4 and S5). Compound 13a was then
obtained by the nucleophilic substitution reaction of 6-chloro-
4
4. RESULTS AND DISCUSSION
2
-fluoropurine with methyl 3-bromopropionate and DIBAL-H
4
.1. In Vitro Anticancer Activities. At first, all
(diisobutyl aluminum hydride)-mediated reduction of the
compounds were tested using their racemic mixtures against
the HCT-116 and SW480 human colon cancer cell lines, using
nucleophilic product giving the yield of 65.3%. Compound 16b
was obtained in 83.5% yield through the deprotection of the
silyl ether group in compound 15b by tetrabutylammonium
fluoride (TBAF). Compounds g (4g−11g) or h (5h−11h)
series were obtained by the nucleophilic substitution reaction
of b or c series with ammonia in MeOH in the yields of 67.5−
5
1
8
-fluorocrail (5-FU) as the positive control. As shown in Table
, compounds 1a−13a, 6b−11b, 4c−9c, 7d−11d, 7e−11e,
f−11f, 8g−11g, 8h−11h, and 5i−9i exhibited moderate to
potent antitumor activities against both cell lines.
1
3
4
In compound a series (R = F, R = Cl, R = H), all of the
7
5.9% (Scheme 1). Finally, compounds 4b−6b were
2
acyclonucleosides with R as H (13a) or C1−C9 linear alkyl
dechlorinated by Pd/C-mediated hydrogenolysis in methanol
to generate 4j−6j in 63.1−66.5% yield. Compounds 5k−10k
were then obtained by introducing an olefin double bond in
the side chain of 6-chloropurine from MBH adducts through
allyl amination in 36.8−43.2% yield (Scheme 1).
The stereochemistry of a chiral compound could usually
affect its bioactivities obviously. To further elucidate the
impact of stereochemistry on the anticancer activity of these
novel ANCs, several chiral acyclonucleosides with (R)-
configuration were generated in 51.3−56.2% overall yield
and 91−95% ee from 6-chloro-9H-purine and several acroleins
using (R)-prolinol-derived chiral secondary amine II as the
(
1a−9a) showed anticancer activities (IC = 7.44−39.81 μM)
50
2
and the R on the 1′ position has a mild but regular impact on
their anticancer activities, which first increased and then
decreased gradually with the increase of the 1′ position alkyl
chain length, with n-amyl (5a) being found to be optimal
against cell lines HCT-116 and SW480 (IC50 = 8.66, 7.44 μM).
With the introduction of heteroatoms and a phenyl ring to the
2
1′-position of the purine side chain, i.e., R = CH
OBn (10a),
2
CH OBz (11a), and CH OTBDMS (12a), their anticancer
2
2
activities (IC = 5.17−8.02 μM) were comparable to those of
5
0
5a.
2
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Table 1. Anticancer Activities of Acyclic Nucleoside Compounds
a
IC50 (μM)
R1
R2
R3
R4
HCT-116
b
SW480
b
compound
1a
2a
3a
4a
F
F
F
F
Me
Et
n-C H
Cl
Cl
Cl
Cl
H
H
H
H
19.76 ± 1.46
18.85 ± 1.22
16.47 ± 3.03
13.46 ± 2.33
16.83 ± 1.09
19.84 ± 1.11
17.19 ± 1.69
13.18 ± 2.01
3
7
n-C H
4
9
5a
6a
7a
8a
9a
F
F
F
F
F
n-C H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
8.66 ± 1.48
9.96 ± 0.72
17.57 ± 3.96
24.22 ± 4.17
32.05 ± 2.42
7.44 ± 1.25
12.94 ± 1.39
26.12 ± 2.26
14.98 ± 0.80
12.46 ± 2.06
5
11
13
15
17
19
n-C H
6
n-C H
7
n-C H
8
n-C H
9
1
1
1
1
1
2
3
4
0a
1a
2a
3a
b
b
b
b
F
F
F
F
H
H
H
H
CH OBn
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
5.24 ± 0.65
5.17 ± 0.75
7.78 ± 1.21
39.81 ± 3.72
>50
>50
>50
>50
7.17 ± 1.25
7.24 ± 0.92
8.02 ± 0.75
29.70 ± 2.95
>50
>50
>50
>50
2
CH OBz
2
CH OTBDMS
2
H
Me
Et
n-C H
3
7
n-C H
4
9
5
6
7
8
9
1
1
1
b
H
H
H
H
H
H
H
H
n-C H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
>50
>50
5
11
13
15
17
19
b
n-C H
11.94 ± 1.17
4.16 ± 2.39
1.65 ± 0.43
0.89 ± 0.21
4.93 ± 0.69
7.59 ± 0.62
>50
16.74 ± 2.12
3.63 ± 2.37
1.37 ± 0.36
1.15 ± 0.31
3.16 ± 0.36
5.29 ± 0.90
>50
6
b
n-C H
7
b
n-C H
8
b
n-C H
9
0b
1b
2b
n-C H
10
21
23
25
n-C H
1
1
n-C H
1
2
13b
14b
15b
16b
4c
H
H
H
H
Cl
CH OBn
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
>50
>50
>50
>50
>50
>50
31.70 ± 4.14
>50
30.90 ± 1.39
2
CH OBz
2
CH OTBDMS
2
CH OH
2
n-C H
15.49 ± 2.01
4
9
5
6
7
8
9
4
5
6
7
8
9
1
1
5
6
7
c
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
n-C H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6.17 ± 0.89
12.10 ± 1.68
12.79 ± 1.75
22.15 ± 3.15
32.56 ± 3.12
>50
6.01 ± 0.85
12.42 ± 1.80
13.56 ± 3.25
24.04 ± 2.49
36.30 ± 1.75
>50
5
11
13
15
17
19
9
c
n-C H
6
c
n-C H
7
c
n-C H
8
c
n-C H
9
d
d
d
d
d
d
0d
1d
e
n-C H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
NHCH3
NHCH3
NHCH3
4
n-C H
>50
>50
5
11
13
15
17
19
n-C H
>50
>50
6
n-C H
15.28 ± 2.53
12.77 ± 4.15
2.53 ± 0.25
7.53 ± 0.83
8.23 ± 2.18
>50
23.92 ± 2.16
24.55 ± 4.37
3.25 ± 0.30
8.26 ± 0.86
9.70 ± 1.41
>50
7
n-C H
8
n-C H
9
n-C H
1
0
21
23
n-C H
1
1
n-C H
5
11
e
n-C H
>50
>50
6
13
e
n-C H
12.53 ± 3.01
15.26 ± 4.28
7
15
2
081
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Table 1. continued
a
IC50 (μM)
compound
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
R2
R3
NHCH3
NHCH3
NHCH3
NHCH3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
NH2
R4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
H
H
H
HCT-116
b
SW480
b
8
9
1
1
5
6
7
8
9
1
1
4
5
6
7
8
9
1
1
5
6
7
8
9
1
1
5
6
7
8
9
4
5
6
5
e
n-C H
3.73 ± 0.47
5.41 ± 1.03
12.70 ± 1.43
15.38 ± 2.29
>50
3.77 ± 0.71
4.45 ± 0.91
11.99 ± 1.97
12.79 ± 2.39
>50
8
17
e
n-C H
9
19
0e
1e
f
n-C H
10
21
23
n-C H
1
1
n-C H
5
11
f
n-C H
>50
>50
6
13
15
17
19
f
n-C H
>50
>50
7
f
n-C H
19.84 ± 2.67
9.81 ± 0.66
5.49 ± 1.69
12.03 ± 0.99
>50
21.61 ± 1.71
12.24 ± 1.33
3.32 ± 0.69
11.75 ± 1.00
>50
8
f
n-C H
9
0f
1f
g
n-C H
10
21
23
n-C H
1
1
n-C H
4
9
g
n-C H
NH2
>50
>50
5
11
13
15
17
19
g
n-C H
NH2
>50
>50
6
g
n-C H
NH2
>50
>50
7
g
n-C H
NH2
11.55 ± 1.81
10.54 ± 2.66
2.59 ± 0.41
13.35 ± 2.66
>50
20.68 ± 1.47
6.03 ± 1.62
1.20 ± 0.17
11.61 ± 0.95
>50
8
g
n-C H
NH2
9
0g
1g
h
h
h
h
h
0h
1h
i
n-C H
NH2
1
0
21
23
n-C H
NH2
1
1
n-C H
NH2
5
11
n-C H
NH2
>50
>50
6
13
15
17
19
n-C H
NH2
>50
>50
7
n-C H
NH2
20.99 ± 2.76
14.84 ± 3.19
11.83 ± 2.00
19.95 ± 2.05
18.29 ± 1.33
16.12 ± 1.15
16.22 ± 1.69
22.39 ± 2.07
26.91 ± 3.79
>50
13.14 ± 2.02
12.22 ± 2.30
13.13 ± 1.24
17.78 ± 1.08
23.81 ± 1.34
14.13 ± 1.01
14.44 ± 1.52
17.78 ± 1.13
24.98 ± 1.76
>50
8
n-C H
NH2
9
n-C H
NH2
1
0
21
23
n-C H
NH2
1
1
n-C H
Cl
5
11
i
n-C H
Cl
6
13
15
17
19
9
i
n-C H
Cl
7
i
n-C H
Cl
8
i
n-C H
Cl
9
j
n-C H
H
4
j
n-C H
H
>50
>50
5
11
j
n-C H
H
>50
>50
6
13
-FU
10.81 ± 0.69
9.70 ± 1.22
a
The antiproliferation activities of individual compounds against tumor cells were determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
b
tetrazolium bromide (MTT) assay. Data are mean ± standard error of the mean (SEM) values from three independent experiments. Both HCT-
1
16 and SW480 are human colon carcinoma cell lines.
1
4
3
Similarly, in compound b series (R = R = H, R = Cl),
9c showed similar anticancer activities (IC50 = 6.01−36.30
μM) to those of 4a−9a (IC = 7.44−32.05 μM), and also the
acyclonucleosides 1b−12b also showed a similar trend in
anticancer activities, which first increased and then decreased
gradually as the 1′-position alkyl chain length increases.
Compound 9b, with the 1′-position of the purine side chain
substituted by n-nonyl, exhibited the highest anticancer
activities against cell lines HCT-116 and SW480 (IC50
.89, 1.15 μM), which were about 10-fold more potent than
5
0
length of alkyl chains on the 1′-position of 4c−9c has a similar
impact on their anticancer activities, which first increased and
then decreased gradually with the increase of 1′-position alkyl
chains.
1
4
3
=
In compound d, e, f, and g series (R = R = H, R = OMe
(d), NHCH (e), N(CH ) (f), and NH (g), respectively),
0
3
3 2
2
those of the positive control 5-FU (IC50 =2 10.81, 9.70 μM).
The anticancer activities were lost when R was replaced with
CH OBn (13b), CH OBz (14b), CH OTBDMS (15b), and
the data pointed toward the same trend that all 4d−11d, 5e−
11e, 5f−11f, and 4g−11g displayed anticancer activities that
first increased and then decreased gradually as the 1′-position
alkyl chain length increases. Compound 10g, with the 1′-
position of the purine side chain substituted by n-decyl,
exhibited the highest anticancer activities against cell lines
2
2
2
CH OH (16b).
2
1
3
4
1
In compound c series (R = R = Cl, R = H), R of a series
was changed from F to Cl, the resulting acyclonucleosides 4c−
2
082
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HCT-116 and SW480 (IC50 = 2.59, 1.20 μM), which were
about 4−10-fold more potent than those of the positive
control 5-FU (IC = 10.81, 9.70 μM). In compound h series
Table 3. Inhibitory Effect of Compounds 5k−10k on Cancer
Cell Proliferation
50
1
3
4
(
R = Cl, R = NH , R = H), acyclonucleosides 5h−11h also
2
continued the same tendency as those of d, e, f, and g series.
With the introduction of bromine to the 8-position of the
1
3
4
purine skeleton (compound i series, R = H, R = Cl, R = Br),
all of the resulting acyclonucleosides 5i−9i showed moderate
and not much different anticancer activities with IC values in
the range of 14.13−26.91 μM. The length of alkyl chains on
the 1′-position of 5i−9i also has a slight impact on their
anticancer activities, which first increased and then decreased
gradually with the increase of 1′-position alkyl chains, with n-
hexyl (6i) and n-heptyl (7i) being found optimal against cell
lines HCT-116 and SW480 (IC50 = 14.13−16.22 μM). When
5
0
a
IC50 (μM)
b
b
compound
R2
HCT-116
>50
SW480
>50
5
k
n-C H
5
11
13
15
17
19
6k
7k
n-C
H
30.21 ± 2.35
22.39 ± 2.18
19.43 ± 2.06
>50
32.03 ± 2.08
29.42 ± 2.01
16.22 ± 1.29
27.61 ± 1.90
6
n-C H
7
2
-, 6-, and 8-positions of the purine skeleton were non-
8
9
k
k
n-C H
8
substituted (compound j series, R = R3 = R = H),
acyclonucleosides 4j−6j exhibited no antitumor activity,
which implied that the 1−2 substituents like halogens on the
purine skeleton contributed greatly to their bioactivities.
Next, some selected compounds (5c, 10g, 11a, 9b, 10b, and
1
4
n-C H
9
1
5
0k
-FU
Ph
33.33 ± 3.13
10.81 ± 0.69
12.91 ± 3.97
9.70 ± 1.22
a
The antiproliferation activities of individual compounds against
tumor cells were determined by the MTT assay. Data are mean ±
SEM values from three independent experiments. Both HCT-116
9
e) were further tested against other cancer cells including
b
HeLa, A549, and U87MG, and all of them showed good
antitumor activities, whereas 5-FU was inactive with the IC >
and SW480 are human colon carcinoma cell lines.
5
0
5
0 μM (Table S1). To clarify the impact of stereochemistry on
anticancer activity, both (R)- and (S)-configured chiral acyclic
nucleosides (7b−9b) were synthesized asymmetrically and
assessed by an MTT assay in this regard (Table 2). All of the
5
k−10k with somewhat decreased anticancer activities
compared with those of corresponding compound b series.
Using the hydroxyl group as a handle, simple synthetic
manipulation of the promising compound 9b readily converted
Table 2. Inhibitory Effects of Compounds 7b−9b (rac-, R-,
and S-) on Cancer Cell Proliferation
it into its OMs, N , triazole, Cl, and phosphonoethyl
derivatives (9ba−9be) in 63.8−75.6% yields and 90−94% ee.
Furthermore, the diethylphosphonate function of compound
3
9
b could also be deprotected to generate the corresponding
phosphonic acid 9bf in 44.9% yield (Scheme 3).
As shown in Table 4, when the hydroxyl group on the side
chain of 9b was substituted by several other types of functional
groups, all of the derivatives (9ba−9be) exerted moderate to
potent anticancer activities (IC = 1.04−13.49 μM) and the
5
0
a
IC50 (μM)
azide-substituted 9bb was found optimal to exhibit the same
level of activities as those of 9b. However, the phosphonic acid
R2
b
b
compound
configuration
HCT-116
SW480
9
bf had almost no antitumor activity. By comparing the
7
8
9
5
b
n-C H
rac
R
S
rac
R
S
rac
R
S
4.16 ± 1.39
2.67 ± 0.37
10.72 ± 0.91
1.65 ± 0.43
1.12 ± 0.28
3.40 ± 0.64
0.89 ± 0.21
0.45 ± 0.10
1.05 ± 0.16
10.81 ± 0.69
3.63 ± 1.37
2.93 ± 0.38
3.82 ± 0.97
1.37 ± 0.36
0.32 ± 0.25
2.33 ± 0.40
1.15 ± 0.31
1.63 ± 0.35
3.62 ± 0.66
9.70 ± 1.22
7
15
17
19
antitumor activities of acyclic nucleoside 9b and phosphonic
acid 9bf, it could be seen that the hydroxyl group in the side
chain of 9b does not form the corresponding nucleoside
triphosphate.
Based on the step-by-step investigation of structure−activity
relationships, compound 9b was identified as the most active
compound possessing potent in vitro anticancer activity against
colon cancer cell lines HCT-116 and SW480 with the IC50
values of 0.89 and 1.15 μM, respectively, and therefore was
selected for further bioactivity evaluation.
b
n-C H
8
b
n-C H
9
-FU
a
The antiproliferation activities of individual compounds against
tumor cells were determined by the MTT assay. Data are mean ±
SEM values from three independent experiments. Both HCT-116
4.2. Toxicity against Human Colon Epithelial Cell. The
b
toxicity of compound 9b against normal cell lines was
evaluated using the normal colon epithelial cell line
NCM460 with 5-FU as the positive control. As shown in
Figure 3, there was no significance between compound 9b and
and SW480 are human colon carcinoma cell lines.
5
-FU on toxicity against the human colon epithelial cell line
(R)-configured acyclonucleoside derivatives showed several
NCM460 at 2, 10, and 50 μM.
fold better anticancer activity than their corresponding
antipodes, indicating that the (R)-configuration of the chiral
center benefited the antiproliferative activity.
As shown in Table 3, the introduction of an olefin double
bond to the side chain of purines resulted in acyclonucleosides
4.3. Apoptosis Induced by Compound 9b. A slight G0/
G1 phase arrest was caused by representative compound 9b at
37
16 μM (SI, Figure S1), prompting us to explore further the
cell apoptosis induced by this compound. In this analysis,
colon cancer cells were treated with vehicle or a solution of
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Scheme 3. Syntheses of Compounds 9ba−9bf
b
Table 4. Inhibitory Effect of Compounds 9ba−9be on Cancer Cell Proliferation
a
The antiproliferation activities of individual compounds against tumor cells were determined by the MTT assay. Data are mean ± SEM values
b
from three independent experiments. Both HCT-116 and SW480 are human colon carcinoma cell lines.
compound 9b at different concentrations (2, 4, 8, 16, or 32
μM) for 48 h sequentially and then stained with Annexin V-
fluorescein isothiocyanate (FITC) and propidium iodide (PI)
in a dark environment. The percentages of apoptotic cells were
manner, and the percentage of apoptotic cells increased from 6
to 69% in SW480.
The apoptosis induced by compound 9b in HCT-116 cells is
shown in Figure S2 (SI). As the concentration of 9b increased,
the proportion of apoptotic cells also increased steadily from
12 to 66%. The results suggest that compound 9b could
38,39
determined by flow cytometry.
As shown in Figure 4,
compound 9b induced apoptosis in a concentration-dependent
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the increasing concentrations of compound 9b (Figure 6A).
Furthermore, flow cytometry results showed that the number
of treated cells emitting red fluorescence decreased signifi-
cantly, while the number of treated cells emitting green
fluorescence obviously increased, indicating considerable
dissipation of MMP (Figure 6B). The results suggest that
the dissipation of MMP may participate in the apoptosis
induced by compound 9b. However, treatment of SW480 cells
46
for 48 h with compound 9b and Q-VD-OPh, a pan-caspase
inhibitor, led only to a negligible change in the proportion of
apoptotic cells, suggesting that cell apoptosis induced by
compound 9b did not occur through the caspase signaling
pathway (SI, Figure S3).
Figure 3. Toxicity of compound 9b on NCM460.
4.6. Inhibition of Colony Formation by Compound
b. The effect of compound 9b on the colony formation of
9
HCT-116 and SW480 was also evaluated. Cells were treated
with a solution of compound 9b at various concentrations for
effectively induce apoptosis in cancer cells in a dose-dependent
manner.
48 h and allowed to grow for another 14 days in a regular
4
.4. Analysis of the Expression of Apoptosis-Related
culture medium, and digital images were taken. As shown in
Figure 7, the exposure of HCT-116 and SW480 cells to the
solutions of compound 9b at low concentrations resulted in a
significant inhibition of colony formation. Cancer cell growth
was completely abolished at concentrations of 16 μM and
above.
Proteins (Bax, P53, Bcl-xl, and Cleaved-PARP) Induced
by Compound 9b. To probe further into the apoptosis
induced by compound 9b, Bax, P53, Bcl-xl, and Cleaved-PARP
were determined with western blotting as the markers of
4
0−44
apoptosis.
As shown in Figure 5, along with the increasing
concentrations of compound 9b, the expressions of Bax, P53,
and Cleaved-PARP were also found to increase gradually,
whereas the expression of Bcl-xl decreased gradually,
suggesting that compound 9b could induce apoptosis.
4.7. Metabolic Stability In Vitro. Metabolic stability of
compound 9b in human and mouse liver microsome was
tested. As shown in Table 5, the t1/2 of compound 9b in human
liver microsome was 10 min, which is similar to that of 5-FU in
4
.5. Mitochondrial Membrane Potential (MMP)
Assay. The loss of mitochondrial membrane potential
MMP) is considered to be a limiting factor in the apoptotic
47
the literature.
.8. Acute Toxicity of Compound 9b. An acute toxicity
4
(
assay was performed to assess the safety of compound 9b in
vivo. As summarized in Table 6, the LD50 value of compound
pathway, which has been regarded as a target for antitumor
agents and hence is of high value in controlling cell
4
5
9
b was 220 mg/kg, which is similar to that of 5-FU in the
apoptosis. To investigate the role of mitochondria in the
apoptosis induced by compound 9b, SW480 cells were stained
with JC-1, and the effect of compound 9b on MMP was
measured. JC-1 is capable of selectively entering mitochondria,
either to form monomers that emit green fluorescence at low
MMP or to form aggregates that emit red fluorescence at high
MMP. Images acquired using fluorescence microscopy showed
that the green area (low MMP) was significantly enlarged with
47
literature.
4.9. Inhibition Activities of 9b on Tumor Growth in a
Xenograft Mice Model. The in vivo antitumor activities of
compound 9b were evaluated on the SW480 xenograft mice
48
model. As shown in Figure 8, eight BALB/C mice per group
were administered via intraperitoneal injection with vehicle,
compound 9b (at dosages of 25 and 50 mg/kg), or 5-FU at a
Figure 4. Induction of apoptosis on SW480 cells by compound 9b. Flow cytometry analysis of apoptotic SW480 cells induced by compound 9b at
different concentrations. Cells were treated with vehicle or compound 9b at 2, 4, 8, 16, and 32 μM for 48 h.
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Figure 5. (A) Western blotting analysis of cell-apoptosis-related proteins after treatment with compound 9b at different concentrations. (B)
Relative amount of protein after treatment with compound 9b at different concentrations.
Figure 6. Collapse of the mitochondrial membrane potential on SW480 cells treated by compound 9b. (A) Changes in the mitochondrial
membrane potential were observed using the JC-1 kit under fluorescence microscopy. (B) Flow cytometry was used to detect the mitochondrial
membrane potential on SW480 cells.
dosage of 50 mg/kg, once every 2 days over a period of 12
days. As shown in Figure 8A−D, compound 9b significantly
inhibited tumor growth in a dose-dependent manner as
measured by tumor weight and volume. At the same time,
the body weight of the treated mice did not show any
significant decrease, suggesting that this compound may have
favorable toxicity properties. As shown in Figure 8E, H & E
staining of the tumors showed that the tumors were
significantly necrotic after the administration of compound 9b.
compounds 21 and 23 showed an inhibitory effect on cancer
cell proliferation to some extent.
The biotin-annexed compounds 21 and the diazirine-based
probe 23 were tested as noncovalent and covalent binding
probes, respectively, to explore the interaction between the
acyclic nucleoside moiety and the target proteins. Probes were
incubated with SW480 cell lysate, and the linkers were used as
negative controls. After incubating overnight, the proteins
bounded to probes were pulled down by streptavidin-
conjugated beads. The 23- and 22-treated samples were
separated with sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to a poly(vinylidene
fluoride) (PVDF) member, and incubated with avidin-HRP
antibody, which can bind to biotin. As shown in Figure 9A, the
4
.10. Fishing for Targets. To identify the targets of these
compounds, the biotin-annexed probe 21 and the diazirine-
based probe 23 were synthesized and their antitumor activities
49,50
were tested in vitro (Schemes 4, 5, and Table 7).
Both
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Figure 7. Inhibitory effects of compound 9b on colony formation of HCT-116 cells and SW480 cells.
Table 5. Metabolic Stability of Compound 9b In Vitro
with an (R)-configuration showed anticancer activities several
folds better than those of their (S)-enantiomers, and the (R)-
a
b
species
T1/2 (min)
clint in vitro (mL/min/g protein)
9
b showed remarkable anticancer activity against colon cancer
human
mouse
10.8
1.00
195
2106
cells HCT-116 with an IC50 value of 0.45 μM.
Compound 9b was found to arrest the cell cycle slightly at
the G0/G1 phase. Cell apoptosis was induced in the presence
of compound 9b, as evidenced by the significant increment on
the expression of proapoptosis proteins Bax and P53, as well as
the cleavage fragment of PARP. On the other hand, the
antiapoptosis protein Bcl-xl was found to attenuate substan-
tially upon treatment with compound 9b. Further mechanistic
studies suggested that the dissipation of MMP may participate
in the apoptosis induced by compound 9b, while the caspase
signaling pathway inhibitor Q-VD-OPh may have little effect
on cell apoptosis. Compound 9b was also shown to have a
long-term toxicity, in the sense that it can effectively inhibit the
colony formation. Moreover, in vivo studies showed that
compound 9b could inhibit tumor growth and induce tumor
necrosis without affecting the body weight of mice, indicating a
potent anticancer activity and low toxicity. The LD50 value and
metabolic stability in vitro of compound 9b is similar to that of
5-FU.
a
b
T1/2: Half-life time. Clint in vitro: Intrinsic clearance in vitro.
compound-23-treated group exhibited a distinct band at 75
Kd, while the linker-22-treated and input groups did not show
any obvious band. Then, both biotin-annexed compound 21
and the diazirine-based probe 23 were separated with SDS-
PAGE and stained by silver staining. The results are shown in
Figure 9B,C, which are in agreement with the western blotting
results, where obvious bands were discovered at 75 Kd. The
silver-stained bands were separated for protein mass
spectrometry. Protein mass spectrometry for the 22-treated
band and the 23-treated band revealed that nucleolin, ezrin,
ATP-dependent RNA helicase, or ATP-binding cassette
protein might be the targets of these molecules. The target
validation is underway in this lab.
5
. CONCLUSIONS
As an ongoing effort to develop promising drug candidates,
we are currently working to decipher the cellular targets of the
anticancer ANCs reported herein. The biotin-annexed probe
and the diazirine-based probe were synthesized to identify the
targets of these compounds, and an obvious band was
discovered at 75 Kd. The bands were separated, and the
protein mass spectrometry revealed that nucleolin, ezrin, ATP-
dependent RNA helicase, or ATP-binding cassette protein
In summary, a series of novel acyclic nucleoside compounds
were designed, synthesized, and evaluated for their anticancer
activities in vitro and in vivo. Structure−activity relationship
studies revealed that many of the target compounds
demonstrated in vitro anticancer activities, among which 9b
was discovered as the most active compound against colon
cancer cells HCT-116 and SW480 with IC values of 0.89 and
50
1
.15 μM, respectively. All chiral acyclic nucleoside derivatives
Table 6. Acute Toxicity of Compound 9b on Balb/C Mice
number of dead mice
day 2 day 3
group
day 1
days 4−15
total death
10
survival (%)
0
300 mg/kg
250 mg/kg
200 mg/kg
150 mg/kg
100 mg/kg
female
male
female
male
female
male
female
male
female
male
female
male
5
5
3
2
3
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
3
0
0
0
20
70
100
100
100
vehicle
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Figure 8. In vivo anticancer activity of compound 9b on the SW480 xenograft mice model. Female BALB/C nude mice were administered by
intraperitoneal injection with the vehicle, compound 9b, at dosages of 25 and 50 mg/kg, or 5-FU at a dosage of 50 mg/kg (n = 8 each group), once
every 2 days for 12 days. Average tumor weight (A), average body weight (B), average tumor volume (C), and the isolated tumors (D) were
measured. Data are expressed as the mean ± standard error (n = 8). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 vs
control. H & E staining analysis of tumors in nude mice on the SW480 xenograft mice model (E).
Scheme 4. Synthesis of the Biotin-Annexed Probe 21
might be the targets of these molecules. The target validation is
underway in this lab.
From a groundbreaking point of view, acyclic nucleoside
compound 9b has demonstrated remarkable potential to be
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Scheme 5. Synthesis of the Diazirine-Based Probe 23
Table 7. Inhibitory Effect of Biotin-Annexed Probe 21 and
Diazirine Photoaffinity Probe 23 on Cancer Cell
Proliferation
anticancer agents and is of great significance in the develop-
ment of more efficient agents for cancer therapy in the future.
a
6. EXPERIMENTAL SECTION
IC50 (μM) ± SEM
b
b
6.1. General Methods for Chemistry. The purities of all
targeted compounds were confirmed to be >95% by high-performance
liquid chromatography (HPLC) (SI). All commercial reagents and
solvents were purchased from commercial suppliers and used without
compound
HCT-116
SW480
2
2
5
1
3
-FU
44.74 ± 4.22
21.49 ± 2.34
10.81 ± 0.69
45.23 ± 3.26
13.00 ± 1.65
9.70 ± 1.22
1
13
further purification. H and C NMR spectral data were recorded
a
The antiproliferation activities of individual compounds against
with a Bruker 400 or 600 MHz spectrometer in CDCl , CD OD,
3
3
tumor cells were determined by the MTT assay. Data are mean ±
SEM values from three independent experiments. Both HCT-116
and SW480 are human colon carcinoma cell lines.
D O, or DMSO-d using tetramethylsilane (TMS) as the internal
2
6
b
standard. Optical rotation was recorded on the Autopol polarimeter.
All products were further characterized by high-resolution mass
spectrometry (HRMS). HRMS was recorded on an ABI/Sciex QStar
mass spectrometer (ESI). Chiral HPLC analysis was recorded on
Thermo Scientific Dionex Ultimate 3000 and Agilent Technologies
T
D
1
260 Infinity. Optical rotations were reported as follows: [α] (c: = g/
100 mL, in solvent). Optical rotations were recorded on an Autopol
automatic polarimeter. Targeted compounds were analyzed by a
Thermo Scientific UltiMate 3000 HPLC instrument with a UV−
visible detector (5 μm, 4.6 mm × 150 mm Agilent C column).
1
8
6
.1.1. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)butan-1-ol (1a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
0
mL) were added to a reaction tube with a magnetic stirring bar. Then,
crotonaldehyde (24.9 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by thin-layer chromatography (TLC). Then, the solution
was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol) was
4
slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
2
4
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 1a as a white
1
solid in 35.6 mg (72.9% yield). White solid. mp: 109−110 °C. H
NMR (400 MHz, CDCl ) δ 8.15 (s, 1H), 4.98−4.87 (m, 1H), 3.72−
3
3
.63 (m, 1H), 3.44−3.35 (m, 1H), 2.30−2.25 (m, 1H), 2.25−2.11
13
(m, 2H), 1.71 (d, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
1
5
58.1, 155.9, 153.6, 153.5, 152.9, 152.7, 145.1, 145.0, 130.7, 130.6,
8.6, 50.1, 38.3, 20.5; ESI-HRMS (m/z) calcd C H ClFN O (M +
9
11
4
+
H ), 245.0600; found, 245.0601.
.1.2. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)pentan-1-ol (2a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-pent-2-enal (29.3 μL, 0.3 mmol) was added to the reaction
6
0
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
Figure 9. Western blotting and silver staining results of the pull-down
samples. (A) Merger of bright field and chemiluminescence of the
PVDF member. (B) and (C) Silver staining results of the SDS-PAGE
gel.
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
used as a lead for further development of a brand-new class of
anticancer agents. Therefore, this work may open an avenue to
further explore the use of ANCs as new and efficacious
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 2a as a white solid in 38.2 mg (74.0% yield). White solid.
1
mp: 98−100 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.69−
3
2
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4
2
.59 (m, 1H), 3.69−3.61 (m, 1H), 3.37−3.28 (m, 1H), 2.45 (s, 1H),
.28−2.17 (m, 2H), 2.17−2.09 (m, 1H), 2.08−1.96 (m, 1H), 0.83 (t,
(100 MHz, CDCl ) δ 158.0, 155.8, 153.8, 153.6, 152.7, 152.5, 145.78,
3
145.75, 130.50, 130.45, 58.4, 55.0, 36.7, 34.1, 31.2, 25.9, 22.4, 13.9;
13
+
J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ 158.1, 155.9, 153.8,
ESI-HRMS (m/z) calcd C H ClFN O (M + H ), 301.1226; found,
3
13 19
4
1
53.7, 152.8, 152.6, 145.73, 145.7, 130.6, 130.5, 58.5, 56.6, 36.5, 27.4,
301.1235.
+
10.9; ESI-HRMS (m/z) calcd C H ClFN O (M + H ), 259.0756;
6.1.6. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)nonan-1-ol (6a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-non-2-enal (49.7 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
1
0
13
4
found, 259.0762.
6
.1.3. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)hexan-1-ol (3a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-hex-2-enal (34.8 μL, 0.3 mmol) was added to the reaction
0
(
mixture. The reaction was stirred at room temperature for 24 h and
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
detected by TLC. Then, the solution was cooled to 0 °C. After that,
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 6a as a white solid in 49.6 mg (78.9% yield). White solid.
2
4
1
column chromatography on silica gel (PE/EA = 1:1) to give the target
mp: 114−116 °C. H NMR (400 MHz, CDCl ) δ 8.10 (s, 1H), 4.76−
3
compound 3a as a white solid in 38.1 mg (70.0% yield). White solid.
4.66 (m, 1H), 3.67−3.59 (m, 1H), 3.35−3.26 (m, 1H), 2.61 (s, 1H),
2.26−2.15 (m, 2H), 2.15−2.05 (m, 1H), 1.98−1.88 (m, 1H), 1.29−
1
mp: 113−115 °C. H NMR (400 MHz, CDCl ) δ 8.10 (s, 1H), 4.80−
3
1
3
4
.71 (m, 1H), 3.70−3.61 (m, 1H), 3.36−3.27 (m, 1H), 2.27−2.16
1.10 (m, 7H), 1.10−0.96 (m, 1H), 0.79 (t, J = 7.2 Hz, 3H); C NMR
(
1
m, 3H), 2.16−2.07 (m, 1H), 1.97−1.87 (m, 1H), 1.30−1.20 (m,
(100 MHz, CDCl ) δ 158.0, 155.9, 153.8, 153.6, 152.7, 152.5, 145.74,
3
1
3
H), 1.19−1.08 (m, 1H), 0.89 (t, J = 7.2 Hz, 3H); C NMR (100
145.71, 130.53, 130.48, 58.4, 55.0, 36.8, 34.2, 31.5, 28.7, 26.2, 22.5,
+
MHz, CDCl ) δ 158.1, 155.9, 153.8, 153.7, 152.9, 152.7, 145.62,
14.0; ESI-HRMS (m/z) calcd C H ClFN O (M + H ), 315.1382;
3
14 21
4
1
45.59, 130.61, 130.57, 58.5, 54.6, 36.8, 36.2, 19.6, 13.6; ESI-HRMS
found, 315.1383.
+
(m/z) calcd C H ClFN O (M + H ), 273.0913; found, 273.0920.
6.1.7. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)decan-1-ol (7a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-dec-2-enal (54.7 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
11
15
4
6
.1.4. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)heptan-1-ol (4a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-hept-2-enal (39.3 μL, 0.3 mmol) was added to the reaction
0
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
4
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 7a as a white solid in 48.2 mg (73.2% yield). White solid.
1
compound 4a as a white solid in 41.2 mg (72.0% yield). White solid.
mp: 104−107 °C. H NMR (400 MHz, CDCl ) δ 8.13 (s, 1H), 4.77−
3
1
mp: 118−120 °C. H NMR (400 MHz, CDCl ) δ 8.13 (s, 1H), 4.76−
4.67 (m, 1H), 3.67−3.59 (m, 1H), 3.35−3.26 (m, 1H), 2.91 (s, 1H),
2.26−2.15 (m, 2H), 2.15−2.05 (m, 1H), 1.99−1.87 (m, 1H), 1.25−
3
4
2
1
.67 (m, 1H), 3.67−3.60 (m, 1H), 3.35−3.27 (m, 1H), 2.82 (s, 1H),
1
3
.27−2.14 (m, 2H), 2.14−2.06 (m, 1H), 1.99−1.89 (m, 1H), 1.32−
1.13 (m, 9H), 1.10−0.98 (m, 1H), 0.80 (t, J = 7.2 Hz, 3H); C NMR
1
3
.19 (m, 3H), 1.09−0.96 (m, 1H), 0.80 (t, J = 7.2 Hz, 3H); C NMR
(100 MHz, CDCl ) δ 158.1, 155.9, 153.8, 153.7, 152.9, 152.7, 145.64,
3
(
100 MHz, CDCl ) δ 158.1, 156.0, 153.8, 153.7, 152.9, 152.7, 145.61,
145.61, 130.61, 130.56, 58.5, 54.9, 36.9, 34.2, 31.7, 29.1, 26.3, 22.6,
3
+
1
45.58, 130.64, 130.59, 58.6, 54.9, 36.9, 33.9, 28.4, 22.2, 13.9; ESI-
14.1; ESI-HRMS (m/z) calcd C H ClFN O (M + H ), 329.1539;
1
5
23
4
+
HRMS (m/z) calcd C H ClFN O (M + H ), 287.1069; found,
found, 329.1540.
1
2
17
4
2
87.1078.
.1.5. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)octan-1-ol (5a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-oct-2-enal (44.8 μL, 0.3 mmol) was added to the reaction
6.1.8. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)undecan-1-ol (8a). 6-
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
6
0
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
4
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 8a as a white solid in 55.2 mg (80.7% yield). White solid.
1
compound 5a as a white solid in 45.6 mg (76.0% yield). White solid.
mp: 116−118 °C. H NMR (400 MHz, CDCl ) δ 8.10 (s, 1H), 4.76−
3
1
mp: 115−117 °C. H NMR (400 MHz, CDCl ) δ 8.14 (s, 1H), 4.77−
4.67 (m, 1H), 3.68−3.60 (m, 1H), 3.36−3.26 (m, 1H), 2.32 (s, 1H),
2.25−2.15 (m, 2H), 2.15−2.06 (m, 1H), 1.99−1.88 (m, 1H), 1.28−
3
4
2
1
.66 (m, 1H), 3.70−3.59 (m, 1H), 3.35−3.27 (m, 1H), 2.77 (s, 1H),
.27−2.15 (m, 2H), 2.15−2.06 (m, 1H), 1.99−1.87 (m, 1H), 1.26−
1.21 (m, 4H), 1.21−1.15 (m, 7H), 1.11−1.00 (m, 1H), 0.83 (t, J =
1
3
13
.17 (m, 5H), 1.11−1.01 (m, 1H), 0.79 (t, J = 6.8 Hz, 3H); C NMR
6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 158.1, 155.9, 153.8,
3
2
090
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
3
53.7, 152.8, 152.7, 145.7, 145.6, 130.6, 130.5, 58.5, 54.9, 36.8, 34.2,
3.77 (m, 1H), 3.53−3.44 (m, 1H), 2.52−2.42 (m, 1H), 2.39−2.29
1
3
1.8, 29.3, 29.2, 29.1, 26.3, 22.7, 14.1; ESI-HRMS (m/z) calcd
(m, 1H), 2.18 (s, 1H); C NMR (150 MHz, CDCl ) δ 166.0, 157.8,
3
+
C H ClFN O (M + H ), 343.1695; found, 343.1697.
156.3, 153.7, 153.6, 153.0, 152.9, 145.94, 145.92, 133.8, 130.7, 130.6,
16
25
4
6
.1.9. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)dodecan-1-ol (9a). 6-
129.6, 128.9, 128.7, 64.9, 58.1, 53.8, 32.4; ESI-HRMS (m/z) calcd
+
Chloro-2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
C H ClFN O (M + H ), 365.0811; found, 365.0809.
1
6
15
4
3
0
6.1.12. 4-(tert-Butyldimethylsilyl)-3-(6-chloro-2-fluoro-9H-purin-
9-yl)butan-1-ol (12a). 6-Chloro-2-fluoro-9H-purine (34.5 mg, 0.2
mmol), L-proline (2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02
mmol), and methanol (1.5 mL) were added to a reaction tube with a
magnetic stirring bar. Then, (E)-4-((tert-butyldimethylsilyl)oxy)but-
2-enal (60.0 mg, 0.3 mmol) was added to the reaction mixture. The
reaction was stirred at room temperature for 24 h and detected by
(
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
4
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 9a as a white solid in 58.7 mg (82.4% yield). White solid.
times. The combined organic extracts were dried over Na SO and
2 4
1
mp: 113−115 °C. H NMR (400 MHz, CDCl ) δ 8.10 (s, 1H), 4.76−
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 2:1) to give the target
3
4
2
1
6
1
3
.67 (m,1H), 3.68−3.60 (m, 1H), 3.35−3.26 (m, 1H), 2.50 (s, 1H),
.26−2.15 (m, 2H), 2.14−2.06 (m, 1H), 1.98−1.88 (m, 1H), 1.27−
.21 (m, 4H), 1.21−1.14 (m, 9H), 1.10−1.00 (m, 1H), 0.82 (t, J =
compound 12a as a white solid in 58.9 mg (78.8% yield). White solid.
1
mp: 83−85 °C. H NMR (600 MHz, CDCl
) δ 8.26 (s, 1H), 4.92−
3
.8 Hz, 3H); 13C NMR (100 MHz, CDCl ) δ 158.1, 155.9, 153.8,
4.87 (m, 1H), 4.05 (dd, J = 10.8, 5.4 Hz, 1H), 3.91 (dd, J = 10.8, 3.6
Hz, 1H), 3.73−3.69 (m, 1H), 3.47−3.42 (m, 1H), 2.41 (s, 1H),
3
53.6, 152.8, 152.6, 145.72, 145.68, 130.6, 130.5, 58.5, 55.0, 36.8,
4.2, 31.9, 29.5, 29.4, 29.3, 29.1, 26.3, 22.7, 14.1; ESI-HRMS (m/z)
calcd C H ClFN O (M + H ), 357.1852; found, 357.1850.
2.33−2.27 (m, 1H), 2.21−2.14 (m, 1H), 0.80 (s, 9H), −0.06 (d, J =
+
13
11.4 Hz, 6H); C NMR (100 MHz, CDCl
) δ 158.1, 156.0, 153.8,
17
27
4
3
6
.1.10. 4-(Benzyloxy)-3-(6-chloro-2-fluoro-9H-purin-9-yl)butan-
153.6, 152.7, 152.5, 146.4, 146.4, 130.24, 130.19, 64.1, 58.5, 55.3,
1
-ol (10a). 6-Chloro-2- fluoro-9H-purine (34.5 mg, 0.2 mmol), L-
33.0, 25.8, 18.1, −5.5, −5.7; ESI-HRMS (m/z) calcd
+
proline (2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-4-(benzyloxy)but-2-enal (52.8 mg, 0.3 mmol)
was added to the reaction mixture. The reaction was stirred at room
temperature for 24 h and detected by TLC. Then, the solution was
C H ClFN O Si (M + H ), 375.1414; found, 375.1424.
1
5
25
4
2
6.1.13. 3-(6-Chloro-2-fluoro-9H-purin-9-yl)propan-1-ol (13a). 6-
Chloro-2-fluoro-9H-purine (1.55 g, 10 mmol), potassium carbonate
(1.66 g, 12 mmol), and dry N,N-dimethylformamide (DMF) (25.0
mL) were added to a reaction tube with a magnetic stirring bar. The
cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol) was slowly
reaction was stirred at 0 °C for 1 h under a N atmosphere. Then,
4
2
added and the reaction was stirred for 10 min. Then, the reaction was
quenched with a saturated ammonium chloride solution and
concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
methyl 3-bromopropionate (1.3 mL, 12 mmol) was slowly added to
the reaction mixture. The reaction was stirred at room temperature
for 48 h and detected by TLC. Then, water was added to the reaction
and the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
2
4
2
4
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 10a as a
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 3:1) to give the compound methyl 3-(6-
chloro-2-fluoro- 9H-purin-9-yl)propanoate as a pale-yellow solid in
2.08 g (80.6% yield). The solution of methyl 3-(6-chloro-2-fluoro-9H-
purin-9-yl)propanoate (25.8 mg, 0.1 mmol) in dry CH Cl (1.0 mL)
1
white solid in 54.7 mg (78.2% yield). White solid. mp: 96−97 °C. H
NMR (600 MHz, CDCl ) δ 8.24 (s, 1H), 7.25−7.22 (m, 3H), 7.13−
3
7
.10 (m, 2H), 4.98−4.93 (m, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.42 (d,
2
2
J = 12.0 Hz, 1H), 3.96 (dd, J = 10.2, 6.6 Hz, 1H), 3.76 (dd, J = 10.2,
was cooled to −78 °C under N . DIBAL-H (0.3 mL, 1.0 M in toluene,
2
3
2
.6 Hz, 1H), 3.70−3.64 (m, 1H), 3.42−3.36 (m, 1H), 2.47 (s, 1H),
0.3 mmol) was added dropwise at −78 °C. The reaction was stirred at
−78 °C for 20 min and then stirred at room temperature until methyl
3-(6-chloro-2-fluoro-9H-purin-9-yl)propanoate was completely con-
sumed. After that, the reaction was quenched with 10% NaOH
solution. Then, water was added to the residue and the mixture was
extracted with CH Cl three times. The combined organic extracts
1
3
.32−2.26 (m, 1H), 2.20−2.14 (m, 1H); C NMR (150 MHz,
CDCl ) δ 157.6, 156.2, 153.6, 153.5, 152.5, 152.4, 146.6, 146.5, 137.0,
3
1
30.32, 130.29, 128.5, 128.2, 127.9, 73.4, 69.8, 58.3, 54.0, 33.1; ESI-
+
HRMS (m/z) calcd C H ClFN O (M + H ), 351.1019; found,
1
6
17
4
2
351.1020.
2
2
6
.1.11. 2-(6-Chloro-2-fluoro-9H-purin-9-yl)-4-hydroxybutyl Ben-
were dried over Na SO and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (PE/EA = 1:1)
2 4
zoate (11a). 6-Chloro- 2-fluoro-9H-purine (34.5 mg, 0.2 mmol), L-
proline (2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-4-oxobut-2-en-1-yl benzoate (57.0 mg, 0.3
mmol) was added to the reaction mixture. The reaction was stirred at
room temperature for 24 h and detected by TLC. Then, the solution
to give the target compound 13a as a white solid in 18.6 mg (80.9%
1
yield). White solid. mp: 111−112 °C. H NMR (400 MHz, CDCl ) δ
3
8.15 (s, 1H), 4.43 (t, J = 6.8 Hz, 2H), 3.65 (t, J = 5.4 Hz, 2H), 2.24 (s,
1H), 2.18−2.08 (m, 2H); C NMR (100 MHz, CDCl ) δ 158.5,
156.3, 154.0, 153.8, 153.0, 152.8, 146.6, 146.5, 130.5, 130.4, 58.5,
1
3
3
+
was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol) was
41.4, 31.8; ESI-HRMS (m/z) calcd C H ClFN O (M + H ),
4
8
9
4
slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 11a as a
231.0443; found, 231.0442.
6.1.14. 3-(6-Chloro-9H-purin-9-yl)butan-1-ol (1b). 6-Chloro-9H-
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, crotonaldehyde
(24.9 μL, 0.3 mmol) was added to the reaction mixture. The reaction
was stirred at room temperature for 24 h and detected by TLC. Then,
2
4
white solid in 58.7 mg (80.6% yield). White solid. mp: 138−140 °C.
the solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6
4
1
H NMR (400 MHz, CDCl ) δ 8.22 (s, 1H), 7.85 (d, J = 7.6 Hz, 2H),
mmol) was slowly added and the reaction was stirred for 10 min.
Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
3
7
4
.55 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 2H), 5.25−5.16 (m, 1H),
.89 (dd, J = 12.0, 8.0 Hz, 1H), 4.73 (dd, J = 12.0, 4.0 Hz, 1H), 3.85−
2
091
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
concentrated in vacuo. The residue was purified by flash column
2.49 (s, 1H), 2.28−2.07 (m, 3H), 2.05−1.94 (m, 1H), 1.39−1.19 (m,
13
3H), 1.15−1.02 (m, 1H), 0.83 (t, J = 7.6 Hz, 3H); C NMR (150
2
4
MHz, CDCl ) δ 152.2, 151.8, 151.4, 144.5, 131.9, 58.4, 54.1, 37.7,
3
chromatography on silica gel (PE/EA = 1:1) to give the target
34.1, 28.5, 22.3, 13.9; ESI-HRMS (m/z) calcd C H ClN O (M +
H ), 269.1164; found, 269.1166.
1
2
18
4
+
compound 1b as a white solid in 38.0 mg (84.1% yield). White solid.
1
mp: 104−106 °C. H NMR (400 MHz, CDCl ) δ 8.73 (s, 1H), 8.19
6.1.18. 3-(6-Chloro-9H-purin-9-yl)octan-1-ol (5b). 6-Chloro-9H-
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-oct-2-enal (44.8
μL, 0.3 mmol) was added to the reaction mixture. The reaction was
stirred at room temperature for 24 h and detected by TLC. Then, the
3
(
s, 1H), 5.08−4.98 (m, 1H), 3.71−3.59 (m, 1H), 3.33−3.21 (m, 1H),
13
2
.80 (s, 1H), 2.26−2.04 (m, 2H), 1.75 (d, J = 6.8 Hz, 3H); C NMR
(
3
2
150 MHz, CDCl ) δ 151.9, 151.7, 151.4, 144.0, 131.9, 58.4, 49.3,
3
+
9.2, 20.7; ESI-HRMS (m/z) calcd C H ClN O (M + H ),
9 12 4
27.0694; found, 227.0694.
.1.15. 3-(6-Chloro-9H-purin-9-yl)pentan-1-ol (2b). 6-Chloro-9H-
6
solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol)
4
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-pent-2-enal
was slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
(
29.3 μL, 0.3 mmol) was added to the reaction mixture. The reaction
was stirred at room temperature for 24 h and detected by TLC. Then,
2
4
the solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6
4
in vacuo. The residue was purified by flash column chromatography
mmol) was slowly added and the reaction was stirred for 10 min.
Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
on silica gel (PE/EA = 1:1) to give the target compound 5b as a white
1
solid in 45.7 mg (81.1% yield). White solid. mp: 113−114 °C. H
NMR (400 MHz, CDCl ) δ 8.69 (s, 1H), 8.13 (s, 1H), 4.86−4.76 (m,
3
1
H), 3.65−3.57 (m, 1H), 3.26−3.17 (m, 1H), 2.87 (s, 1H), 2.22−
2
4
2.10 (m, 3H), 2.01−1.91 (m, 1H), 1.28−1.16 (m, 5H), 1.12−1.03
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
13
(m, 1H), 0.79 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
1
2
2
52.1, 151.7, 151.2, 144.7, 131.8, 58.3, 54.2, 37.5, 34.3, 31.2, 26.0,
compound 2b as a white solid in 39.4 mg (82.1% yield). White solid.
+
2.4, 14.0; ESI-HRMS (m/z) calcd C H ClN O (M + H ),
1
13 20
4
mp: 100−103 °C. H NMR (400 MHz, CDCl ) δ 8.70 (s, 1H), 8.14
3
83.1320; found, 283.1330.
(
s, 1H), 4.79−4.70 (m, 1H), 3.67−3.60 (m, 1H), 3.28−3.19 (m, 1H),
6
.1.19. 3-(6-Chloro-9H-purin-9-yl)nonan-1-ol (6b). 6-Chloro-9H-
2
.69 (s, 1H), 2.28−2.12 (m, 3H), 2.12−1.99 (m, 1H), 0.85 (t, J = 7.2
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-non-2-enal (49.7
μL, 0.3 mmol) was added to the reaction mixture. The reaction was
stirred at room temperature for 24 h and detected by TLC. Then, the
13
Hz, 3H); C NMR (150 MHz, CDCl ) δ 152.2, 151.8, 151.3, 144.6,
3
1
31.9, 58.4, 55.8, 37.2, 27.6, 11.0; ESI-HRMS (m/z) calcd
+
C H ClN O (M + H ), 241.0851; found, 241.0855.
10
14
4
6
.1.16. 3-(6-Chloro-9H-purin-9-yl)hexan-1-ol (3b). 6-Chloro-9H-
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-hex-2-enal (34.8
μL, 0.3 mmol) was added to the reaction mixture. The reaction was
stirred at room temperature for 24 h and detected by TLC. Then, the
solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol)
4
was slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol)
4
2
4
was slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 6b as a white
1
solid in 49.1 mg (83.2% yield). White solid. mp: 99−100 °C. H
NMR (400 MHz, CDCl ) δ 8.71 (s, 1H), 8.13 (s, 1H), 4.88−4.77 (m,
3
2
4
1
2
H), 3.66−3.58 (m, 1H), 3.26−3.16 (m, 1H), 2.59 (s, 1H), 2.27−
.08 (m, 3H), 2.03−1.93 (m, 1H), 1.29−1.15 (m, 7H), 1.14−1.05
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 3b as a white
1
3
1
(m, 1H), 0.82 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
solid in 40.1 mg (78.9% yield). White solid. mp: 105−107 °C. H
1
2
2
52.2, 151.8, 151.4, 144.5, 131.9, 58.4, 54.1, 37.6, 34.4, 31.6, 28.8,
NMR (400 MHz, CDCl ) δ 8.73 (s, 1H), 8.14 (s, 1H), 4.91−4.81 (m,
3
+
6.3, 22.6, 14.1; ESI-HRMS (m/z) calcd C H ClN O (M + H ),
1
2
H), 3.69−3.58 (m, 1H), 3.20 (t, J = 9.2 Hz, 1H), 2.47 (s, 1H),
.27−2.08 (m, 3H), 2.03−1.91 (m, 1H), 1.34−1.12 (m, 2H), 0.91 (t,
14 22
4
97.1477; found, 297.1479.
6.1.20. 3-(6-Chloro-9H-purin-9-yl)decan-1-ol (7b). 6-Chloro-9H-
13
J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 152.2, 151.8, 151.3,
3
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-dec-2-enal (54.7
μL, 0.3 mmol) was added to the reaction mixture. The reaction was
stirred at room temperature for 24 h and detected by TLC. Then, the
1
44.5, 131.9, 58.4, 53.8, 37.6, 36.4, 19.6, 13.6; ESI-HRMS (m/z)
+
calcd C H ClN O (M + H ), 255.1007; found, 255.1014.
11
16
4
6
.1.17. 3-(6-Chloro-9H-purin-9-yl)heptan-1-ol (4b). 6-Chloro-9H-
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-hept-2-enal
solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol)
4
was slowly added and the reaction was stirred for 10 min. Then, the
reaction was quenched with a saturated ammonium chloride solution
and concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
(
39.3 μL, 0.3 mmol) was added to the reaction mixture. The reaction
was stirred at room temperature for 24 h and detected by TLC. Then,
the solution was cooled to 0 °C. After that, NaBH (22.7 mg, 0.6
mmol) was slowly added and the reaction was stirred for 10 min.
Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
compound 4b as a white solid in 44.0 mg (82.3% yield). White solid.
4
combined organic extracts were dried over Na SO and concentrated
2 4
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 7b as a white
1
solid in 52.8 mg (85.2% yield). White solid. mp: 101−102 °C. H
2
4
NMR (400 MHz, CDCl ) δ 8.69 (s, 1H), 8.13 (s, 1H), 4.86−4.76 (m,
3
1H), 3.65−3.57 (m, 1H), 3.26−3.16 (m, 1H), 2.84 (s, 1H), 2.25−
2.09 (m, 3H), 2.02−1.91 (m, 1H), 1.27−1.12 (m, 9H), 1.10−1.01
1
13
mp: 87−89 °C. H NMR (400 MHz, CDCl ) δ 8.73 (s, 1H), 8.14 (s,
1
(m, 1H), 0.81 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
3
H), 4.88−4.78 (m, 1H), 3.68−3.58 (m, 1H), 3.25−3.15 (m, 1H),
152.1, 151.7, 151.2, 144.6, 131.8, 58.3, 54.2, 37.5, 34.3, 31.7, 29.04,
2
092
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2
9.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z) calcd C H ClN O (M +
compound 8b as a white solid in 51.1 mg (78.8% yield). White solid.
1
5
24
4
+
1
H ), 311.1633; found, 311.1633.
.1.21. (R)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((R)-7b). Tol-
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-dec-2-enal
54.7 μL, 0.3 mmol), and (S)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. II) (11.9 mg,
.02 mmol) were added to a reaction tube with a magnetic stirring
mp: 106−108 °C. H NMR (400 MHz, CDCl ) δ 8.69 (s, 1H), 8.13
3
6
(s, 1H), 4.86−4.76 (m, 1H), 3.66−3.57 (m, 1H), 3.27−3.15 (m, 1H),
2.80 (s, 1H), 2.25−2.10 (m, 3H), 2.02−1.91 (m, 1H), 1.25−1.20 (m,
4H), 1.20−1.14 (m, 7H), 1.12−1.02 (m, 1H), 0.82 (t, J = 7.2 Hz,
(
1
3
3H); C NMR (100 MHz, CDCl ) δ 152.1, 151.8, 151.3, 144.6,
3
0
131.8, 58.3, 54.2, 37.5, 34.4, 31.8, 29.3, 29.2, 29.1, 26.3, 22.7, 14.1;
+
bar. The mixture was stirred for 30 min at room temperature, and
then, 6-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C.
The reaction was stirred at −30 °C for 24 h and detected by TLC.
The resulting mixture was diluted with precooled MeOH (1.0 mL).
ESI-HRMS (m/z) calcd C H ClN O (M + H ), 325.1790; found,
1
6
26
4
325.1795.
6.1.24. (R)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((R)-8b). Tol-
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-undec-2-enal
(59.5 μL, 0.3 mmol), and (S)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. II) (11.9 mg,
0.02 mmol) were added to a reaction tube with a magnetic stirring
bar. The mixture was stirred for 30 min at room temperature, and
then, 6-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C.
The reaction was stirred at −30 °C for 24 h and detected by TLC.
The resulting mixture was diluted with precooled MeOH (1.0 mL).
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound (R)-7b as a white solid in 34.0 mg (54.8% yield). White
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
4
2
0
solid. mp: 101−102 °C. [α] = −8.4 (c = 0.14, CHCl ), 91% ee
stirred for 10 min. Then, the reaction was quenched with saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
D
3
[
CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
min, temperature = 25 °C, λ = 250 nm, retention time: 30.142 min
1
(
minor), 32.460 min (major)]. H NMR (400 MHz, CDCl ) δ 8.69
3
(
s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
2
4
3
1
.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
column chromatography on silica gel (PE/EA = 1:1) to give the target
1
3
.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
compound (R)-8b as a white solid in 34.1 mg (52.6% yield). White
2
0
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
solid. mp: 101−102 °C. [α] = −2.9 (c = 0.14, CHCl ), 91% ee
3
D
3
5
4.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
[CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
+
calcd C H ClN O (M + H ), 311.1633; found, 311.1633.
min, temperature = 25 °C, λ = 250 nm, retention time: 28.368 min
15
24
4
1
6
.1.22. (S)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((S)-7b). Tol-
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-dec-2-enal
54.7 μL, 0.3 mmol), and (R)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. III) (11.9 mg,
.02 mmol) were added to a reaction tube with a magnetic stirring
bar. The mixture was stirred for 30 min at room temperature and then
(minor), 30.165 min (major)]. H NMR (400 MHz, CDCl ) δ 8.69
3
(s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
3.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
(
1
3
1.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
0
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
3
54.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
+
6
-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C. The
calcd C H ClN O (M + H ), 325.1790; found, 325.1795.
1
6
26
4
reaction was stirred at −30 °C for 24 h and detected by TLC. The
resulting mixture was diluted with precooled MeOH (1.0 mL).
6.1.25. (S)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((S)-8b). Tol-
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-undec-2-
enal (59.5 μL, 0.3 mmol), and (R)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. III) (11.9 mg,
0.02 mmol) were added to a reaction tube with a magnetic stirring
bar. The mixture was stirred for 30 min at room temperature, and
then, 6-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C.
The reaction was stirred at −30 °C for 24 h and detected by TLC.
The resulting mixture was diluted with precooled MeOH (1.0 mL).
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound (S)-7b as a white solid in 33.2 mg (53.5% yield). White
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
4
2
0
solid. mp: 101−102 °C. [α] = 8.4 (c = 0.14, CHCl ), 91% ee
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
D
3
[
CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
min, temperature = 25 °C, λ = 250 nm, retention time: 29.500 min
1
(
major), 34.580 min (minor)]. H NMR (400 MHz, CDCl ) δ 8.69
3
(
s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
2
4
3
1
.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
column chromatography on silica gel (PE/EA = 1:1) to give the target
1
3
.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
compound (S)-8b as a white solid in 32.7 mg (50.5% yield). White
2
0
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
solid. mp: 101−102 °C. [α] = 3.0 (c = 0.20, CHCl ), 92% ee
3
D
3
5
4.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
[CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
+
calcd C H ClN O (M + H ), 311.1633; found, 311.1633.
min, temperature = 25 °C, λ = 250 nm, retention time: 28.079 min
15
24
4
1
6
.1.23. 3-(6-Chloro-9H-purin-9-yl)undecan-1-ol (8b). 6-Chloro-
(major), 32.026 min (minor)]. H NMR (400 MHz, CDCl ) δ 8.69
3
9
H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
(s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
3.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-
undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
1
3
1.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
3
54.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
+
calcd C H ClN O (M + H ), 325.1790; found, 325.1795
16
26
4
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
6.1.26. 3-(6-Chloro-9H-purin-9-yl)dodecan-1-ol (9b). 6-Chloro-
9H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-
dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
2
093
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
6.1.29. 3-(6-Chloro-9H-purin-9-yl)tridecan-1-ol (10b). 6-Chloro-
9H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-
tridec-2-enal (58.9 mg, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
(22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (PE/EA = 3:1) to give the target
compound 9b as a white solid in 52.4 mg (77.5% yield). White solid.
mp: 101−102 °C. H NMR (400 MHz, CDCl ) δ 8.69 (s, 1H), 8.17
(
2
4
2
4
1
3
s, 1H), 4.86−4.77 (m, 1H), 3.65−3.57 (m, 1H), 3.26−3.17 (m, 1H),
.93 (s, 1H), 2.23−2.10 (m, 3H), 2.01−1.90 (m, 1H), 1.25−1.20 (m,
H), 1.20−1.14 (m, 9H), 1.11−1.02 (m, 1H), 0.82 (t, J = 6.8 Hz,
3
H); 13C NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.7,
3
2
4
1
31.8, 58.3, 54.2, 37.5, 34.3, 31.9, 29.44, 29.36, 29.2, 29.1, 26.3, 22.7,
column chromatography on silica gel (PE/EA = 1:1) to give the target
+
14.2; ESI-HRMS (m/z) calcd C H ClN O (M + H ), 339.1946;
compound 10b as a white solid in 55.5 mg (78.8% yield). White solid.
1
7
28
4
1
found, 339.1945.
mp: 108−110 °C. H NMR (400 MHz, CDCl ) δ 8.70 (s, 1H), 8.13
3
6
.1.27. (R)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((R)-9b). Tol-
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-dodec-2-enal
64.4 μL, 0.3 mmol), and (S)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. II) (11.9 mg,
.02 mmol) were added to a reaction tube with a magnetic stirring
bar. The mixture was stirred for 30 min at room temperature and then
(s, 1H), 4.86−4.77 (m, 1H), 3.66−3.58 (m, 1H), 3.25−3.17 (m, 1H),
2.65 (s, 1H), 2.26−2.08 (m, 3H), 2.04−1.90 (m, 1H), 1.27−1.22 (m,
4H), 1.22−1.14 (m, 11H), 1.13−1.04 (m, 1H), 0.84 (t, J = 7.2 Hz,
(
1
3
3H); C NMR (100 MHz, CDCl ) δ 152.2, 151.8, 151.4, 144.5,
3
0
131.9, 58.4, 54.1, 37.7, 34.4, 32.0, 29.6, 29.5, 29.4, 29.36, 29.1, 26.4,
+
22.8, 14.2; ESI-HRMS (m/z) calcd C H ClN O (M + H ),
1
8
30
4
6
-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C. The
353.2103; found, 353.2102.
reaction was stirred at −30 °C for 24 h and detected by TLC. The
resulting mixture was diluted with precooled MeOH (1.0 mL).
6.1.30. 3-(6-Chloro-9H-purin-9-yl)tetradecan-1-ol (11b). 6-
Chloro-9H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
tetradec-2-enal (63.1 mg, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound (R)-9b as a white solid in 34.8 mg (51.5% yield). White
2
4
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
0
solid. mp: 101−102 °C. [α] = −5.0 (c = 0.12, CHCl ), 95% ee
D
3
[
CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
min, temperature = 25 °C, λ = 250 nm, retention time: 26.768 min
2
4
1
(
(
minor), 28.771 min (major)]. H NMR (400 MHz, CDCl ) δ 8.69
column chromatography on silica gel (PE/EA = 1:1) to give the target
3
s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
compound 11b as a white solid in 56.7 mg (77.4% yield). White solid.
1
3
1
.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
mp: 91−93 °C. H NMR (400 MHz, CDCl ) δ 8.71 (s, 1H), 8.13 (s,
3
1
3
.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
1H), 4.87−4.78 (m, 1H), 3.66−3.59 (m, 1H), 3.25−3.17 (m, 1H),
2.65 (s, 1H), 2.26−2.08 (m, 3H), 2.02−1.92 (m, 1H), 1.28−1.16 (m,
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
3
13
54.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
17H), 1.12−1.02 (m, 1H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (100
+
calcd C H ClN O (M + H ), 339.1946; found, 339.1945.
MHz, CDCl ) δ 152.2, 151.8, 151.4, 144.5, 131.9, 58.4, 54.1, 37.7,
17
28
4
3
6
.1.28. (S)-3-(6-Chloro-9H-purin-9-yl)decan-1-ol ((S)-9b). Tol-
34.4, 32.0, 29.7, 29.6, 29.5, 29.4, 29.1, 26.4, 22.8, 14.2; ESI-HRMS
+
uene (2.0 mL), benzoic acid (2.4 mg, 0.02 mmol), (E)-dodec-2-
enal (64.4 μL, 0.3 mmol), and (R)-2-[(bis(3,5-bis(trifluoromethyl)-
phenyl)trimethylsilanyloxy)methyl]pyrrolidine (Cat. III) (11.9 mg,
(m/z) calcd C H ClN O (M + H ), 367.2259; found, 367.2261.
1
9
32
4
6.1.31. 3-(6-Chloro-9H-purin-9-yl)pentadecan-1-ol (12b). 6-
Chloro-9H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
pentadec-2-enal (67.3 mg, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
0.02 mmol) were added to a reaction tube with a magnetic stirring
bar. The mixture was stirred for 30 min at room temperature, and
then, 6-chloro-9H-purine (30.9 mg, 0.2 mmol) was added at −30 °C.
The reaction was stirred at −30 °C for 24 h and detected by TLC.
The resulting mixture was diluted with precooled MeOH (1.0 mL).
NaBH (22.7 mg, 0.6 mmol) was slowly added, and the reaction was
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound (S)-9b as a white solid in 33.4 mg (50.9% yield). White
compound 12b as a white solid in 61.6 mg (81.1% yield). White solid.
2
0
1
solid. mp: 101−102 °C. [α] = 5.0 (c = 0.12, CHCl ), 95% ee
mp: 112−114 °C. H NMR (400 MHz, CDCl ) δ 8.69 (s, 1H), 8.12
D
3
3
[
CHIRALCEL ID, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
(s, 1H), 4.86−4.77 (m, 1H), 3.65−3.58 (m, 1H), 3.25−3.17 (m, 1H),
2.77 (s, 1H), 2.26−2.07 (m, 3H), 2.02−1.91 (m, 1H), 1.27−1.15 (m,
min, temperature = 25 °C, λ = 250 nm, retention time: 25.799 min
1
13
(
major), 29.523 min (minor)]. H NMR (400 MHz, CDCl ) δ 8.69
19H), 1.12−1.02 (m, 1H), 0.84 (t, J = 6.8 Hz, 3H). C NMR (100
3
(
s, 1H), 8.13 (s, 1H), 4.86−4.76 (m, 1H), 3.65−3.57 (m, 1H), 3.26−
MHz, CDCl ) δ 152.1, 151.8, 151.3, 144.6, 131.9, 58.4, 54.1, 37.6,
3
3
1
.16 (m, 1H), 2.84 (s, 1H), 2.25−2.09 (m, 3H), 2.02−1.91 (m, 1H),
34.4, 32.0, 29.7, 29.6, 29.5, 29.42, 29.4, 29.1, 26.4, 22.8, 14.2; ESI-
1
3
+
.27−1.12 (m, 9H), 1.10−1.01 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
C
HRMS (m/z) calcd C H ClN O (M + H ), 381.2416; found,
2
0
34
4
NMR (100 MHz, CDCl ) δ 152.1, 151.7, 151.2, 144.6, 131.8, 58.3,
381.2424.
3
5
4.2, 37.5, 34.3, 31.7, 29.04, 29.03, 26.3, 22.6, 14.1; ESI-HRMS (m/z)
6.1.32. 4-(Benzyloxy)-3-(6-chloro-9H-purin-9-yl)butan-1-ol
(13b). 6-Chloro-9H-purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg,
+
calcd C H ClN O (M + H ), 339.1946; found, 339.1945.
17
28
4
2
094
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
0
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-4-(benzyloxy)but-2-enal (52.8 mg, 0.3 mmol) was added to the
mg, 0.2 mmol), and tetrahydrofuran (1.0 mL) were added to a
reaction tube with a magnetic stirring bar. The reaction was stirred at
room temperature for 2 h and detected by TLC. Then, the solution
was concentrated in vacuo. Finally, the residue was purified by flash
column chromatography on silica gel (PE/EA = 1:1) to give the target
(
reaction mixture. The reaction was stirred at room temperature for 24
h and detected by TLC. Then, the solution was cooled to 0 °C. After
that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction
was stirred for 10 min. Then, the reaction was quenched with a
saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
compound 16b as a white solid in 40.4 mg (83.5% yield). White solid.
4
1
mp: 143−144 °C. H NMR (600 MHz, CD OD) δ 8.71 (s, 1H), 8.62
3
(s, 1H), 4.98−4.92 (m, 1H), 4.12 (dd, J = 12.0, 7.8 Hz, 1H), 3.92
(dd, J = 12.0, 4.2 Hz, 1H), 3.62−3.56 (m, 1H), 3.44−3.38 (m, 1H),
1
3
2.42−2.35 (m, 1H), 2.24−2.16 (m, 1H); C NMR (150 MHz,
CD OD) δ 153.5, 152.6, 151.1, 148.3, 132.5, 63.8, 59.0, 58.0, 33.6;
2
4
3
+
purified by flash column chromatography on silica gel (PE/EA = 1:1)
ESI-HRMS (m/z) calcd C H ClN O (M + H ), 243.0643; found,
9
12
4
2
to give the target compound 13b as a colorless oil in 48.0 mg (72.3%
243.0644.
1
yield). Colorless oil. H NMR (400 MHz, CD OD) δ 8.61 (s, 1H),
6.1.36. 3-(2,6-Dichloro-9H-purin-9-yl)heptan-1-ol (4c). 2,6-Di-
chloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
hept-2-enal (39.3 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
3
8
.57 (s, 1H), 7.18−7.14 (m, 3H), 7.06−7.02 (m, 2H), 5.11−5.03 (m,
1H), 4.49 (d, J = 12.0 Hz, 1H), 4.39 (d, J = 12.0 Hz, 1H), 4.03 (dd, J
=
10.4, 8.0 Hz, 1H), 3.83 (dd, J = 10.4, 4.0 Hz, 1H), 3.61−3.54 (m,
1H), 3.44−3.36 (m, 1H), 2.46−2.35 (m, 1H), 2.25−2.14 (m, 1H);
13
C NMR (100 MHz, CDCl ) δ 151.9, 151.6, 151.0, 145.7, 137.0,
3
31.5, 128.6, 128.2, 127.8, 73.5, 70.3, 58.1, 53.3, 33.7; ESI-HRMS
by TLC Then, the solution was cooled to 0 °C. After that, NaBH
4
+
(22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
16
18
4
2
6
2
4
the reaction mixture. The reaction was stirred at room temperature
column chromatography on silica gel (PE/EA = 1:1) to give the target
for 24 h and detected by TLC. Then, the solution was cooled to 0 °C.
compound 4c as a white solid in 45.8 mg (75.8% yield). White solid.
1
After that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the
mp: 143−144 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.80−
4
3
reaction was stirred for 10 min. Then, the reaction was quenched with
a saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
4.71 (m, 1H), 3.69−3.61 (m, 1H), 3.35−3.26 (m, 1H), 2.29−2.16
(m, 3H), 2.16−2.07 (m, 1H), 2.02−1.91 (m, 1H), 1.33−1.23 (m,
13
3H), 1.12−0.99 (m, 1H), 0.83 (t, J = 7.2 Hz, 3H); C NMR (100
MHz, CDCl ) δ 153.4, 152.8, 151.9, 145.5, 131.1, 58.6, 54.8, 37.1,
3
34.0, 28.4, 22.2, 13.9; ESI-HRMS (m/z) calcd C H Cl N O (M +
2
4
12 17
2
4
+
purified by flash column chromatography on silica gel (PE/EA = 1:1)
H ), 303.0774; found, 303.0775.
to give the target compound 14b as a white solid in 56.5 mg (81.7%
6.1.37. 3-(2,6-Dichloro-9H-purin-9-yl)octan-1-ol (5c). 2,6-Di-
chloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
oct-2-enal (44.8 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
(22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
1
yield). White solid. mp: 137−140 °C. H NMR (400 MHz, CDCl ) δ
3
8
7
.69 (s, 1H), 8.28 (s, 1H), 7.84−7.79 (m, 2H), 7.55−7.49 (m, 1H),
.36 (t, J = 8.0 Hz, 2H), 5.33−5.24 (m, 1H), 4.93 (dd, J = 12.0, 8.0
Hz, 1H), 4.75 (dd, J = 12.0, 4.0 Hz, 1H), 3.82−3.73 (m, 1H), 3.47−
3
.37 (m, 1H), 2.92 (s, 1H), 2.51−2.41 (m, 1H), 2.39−2.29 (m, 1H);
1
3
C NMR (100 MHz, CDCl ) δ 166.0, 151.9, 151.3, 145.2, 133.7,
3
1
31.9, 129.6, 129.0, 128.7, 65.0, 57.9, 53.4, 32.8; ESI-HRMS (m/z)
+
calcd C H ClN O (M + H ), 347.0905; found, 347.0914.
16
16
4
3
6
.1.34. 4-(tert-Butyldimethylsilyl)-3-(6-chloro-9H-purin-9-yl)-
butan-1-ol (15b). 6-Chloro-9H-purine (30.9 mg, 0.2 mmol), L-
proline (2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-4-((tert-butyldimethylsilyl)oxy)but-2-enal
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
(
60.0 mg, 0.3 mmol) was added to the reaction mixture. The
compound 5c as a white solid in 52.6 mg (83.2% yield). White solid.
1
reaction was stirred at room temperature for 24 h and detected by
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
mp: 113−115 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.80−
3
4.71 (m, 1H), 3.69−3.59 (m, 1H), 3.36−3.25 (m, 1H), 2.33 (s, 1H),
2.22−2.16 (m, 2H), 2.15−2.06 (m, 1H), 1.99−1.88 (m, 1H), 1.26−
4
1
3
1.18 (m, 5H), 1.12−1.02 (m, 1H), 0.81 (t, J = 6.8 Hz, 3H); C NMR
(100 MHz, CDCl ) δ 153.3, 152.7, 151.8, 145.5, 131.0, 58.5, 54.9,
3
37.0, 34.2, 31.2, 26.0, 22.4, 14.0; ESI-HRMS (m/z) calcd
+
C H Cl N O (M + H ), 317.0930; found, 317.0939.
2
4
13 19
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 2:1) to give the target
compound 15b as a white solid in 51.4 mg (72.2% yield). White solid.
6.1.38. 3-(2,6-Dichloro-9H-purin-9-yl)nonan-1-ol (6c). 2,6-Di-
chloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
non-2-enal (49.7 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
(22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
1
mp: 105−107 °C. H NMR (400 MHz, CDCl ) δ 8.66 (s, 1H), 8.32
3
(
s, 1H), 5.01−4.93 (m, 1H), 4.08 (dd, J = 10.8, 5.6 Hz, 1H), 3.95
dd, J = 10.8, 3.2 Hz, 1H), 3.72−3.63 (m, 1H), 3.40−3.30 (m, 1H),
(
3
−
1
.02 (s, 1H), 2.33−2.23 (m, 1H), 2.21−2.12 (m, 1H), 0.79 (s, 9H),
1
3
0.07 (d, J = 7.6 Hz, 6H); C NMR (100 MHz, CDCl ) δ 152.0,
3
51.6, 151.1, 145.6, 131.5, 64.4, 58.2, 54.8, 33.5, 25.7, 18.1, −5.6,
5.7; ESI-HRMS (m/z) calcd C H ClN O Si (M + H ), 357.1508;
+
−
15 26 4 2
found, 357.1511.
6
.1.35. 2-(6-Chloro-9H-purin-9-yl)butane-1,4-diol (16b). Com-
pound 15b (35.6 mg, 0.1 mmol), tetrabutylammonium fluoride (52.4
2
4
2
095
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
column chromatography on silica gel (PE/EA = 1:1) to give the target
2.25−2.15 (m, 2H), 2.15−2.06 (m, 1H), 1.98−1.89 (m, 1H), 1.25−
compound 6c as a white solid in 51.5 mg (78.1% yield). White solid.
1.19 (m, 5H), 1.19−1.11 (m, 8H), 1.08−1.00 (m, 1H), 0.82 (t, J =
1
13
mp: 118−119 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.80−
7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 153.4, 152.8, 151.9,
3
3
4
2
1
.71 (m, 1H), 3.68−3.60 (m, 1H), 3.35−3.26 (m, 1H), 2.42 (s, 1H),
145.5, 131.0, 58.6, 54.8, 37.0, 34.3, 31.9, 29.5, 29.4, 29.3, 29.1, 26.3,
+
.23−2.16 (m, 2H), 2.16−2.06 (m, 1H), 2.00−1.89 (m, 1H), 1.27−
22.7, 14.2; ESI-HRMS (m/z) calcd C H Cl N O (M + H ),
1
7
27
2
4
1
3
.12 (m, 7H), 1.10−1.00 (m, 1H), 0.81 (t, J = 6.8 Hz, 3H); C NMR
373.1556; found, 373.1561.
(
100 MHz, CDCl ) δ 153.3, 152.7, 151.8, 145.5, 131.0, 58.5, 54.9,
6.1.42. 3-(6-Methoxy-9H-purin-9-yl)heptan-1-ol (4d). 6-Methoxy-
9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-hept-
2-enal (39.3 μL, 0.3 mmol) was added to the reaction mixture. The
reaction was stirred at room temperature for 24 h and detected by
3
3
7.0, 34.3, 31.5, 28.7, 26.2, 22.5, 14.0; ESI-HRMS (m/z) calcd
+
C H Cl N O (M + H ), 331.1087; found, 331.1083.
14
21
2
4
6
.1.39. 3-(2,6-Dichloro-9H-purin-9-yl)decan-1-ol (7c). 2,6-Di-
chloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
dec-2-enal (54.7 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
4
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
compound 4d as a white solid in 43.6 mg (82.2% yield). White solid.
1
mp: 68−71 °C. H NMR (600 MHz, CDCl ) δ 8.51 (s, 1H), 7.94 (s,
2
4
3
column chromatography on silica gel (PE/EA = 1:1) to give the target
1H), 4.83−4.76 (m, 1H), 4.19 (s, 3H), 3.61−3.55 (m, 1H), 3.27 (s,
1H), 3.12 (td, J = 10.8, 3.6 Hz, 1H), 2.23−2.16 (m, 1H), 2.16−2.09
(m,1H), 2.01−1.91 (m, 2H), 1.35−1.25 (m, 3H), 1.18−1.10 (m,
compound 7c as a white solid in 52.7 mg (76.6% yield). White solid.
1
mp: 111−114 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.80−
3
1
3
4
2
1
.70 (m, 1H), 3.69−3.59 (m, 1H), 3.36−3.26 (m, 1H), 2.40 (s, 1H),
1H), 0.84 (t, J = 6.6 Hz, 3H); C NMR (150 MHz, CDCl ) δ 161.4,
3
.23−2.15 (m, 2H), 2.15−2.06 (m, 1H), 2.00−1.89 (m, 1H), 1.26−
152.5, 152.1, 140.8, 121.5, 58.2, 54.5, 52.7, 38.7, 34.2, 28.6, 22.3, 14.0;
1
3
+
.12 (m, 9H), 1.11−0.99 (m, 1H), 0.82 (t, J = 6.8 Hz, 3H); C NMR
ESI-HRMS (m/z) calcd C H N O (M + H ), 265.1659; found,
1
3
21
4
2
(
100 MHz, CDCl ) δ 153.3, 152.7, 151.8, 145.5, 131.0, 58.5, 54.9,
265.1660.
3
3
7.0, 34.3, 31.7, 29.0, 26.3, 22.6, 14.1; ESI-HRMS (m/z) calcd
6.1.43. 3-(6-Methoxy-9H-purin-9-yl)octan-1-ol (5d). 6-Methoxy-
9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-oct-2-
enal (44.8 μL, 0.3 mmol) was added to the reaction mixture. The
reaction was stirred at room temperature for 24 h and detected by
+
C H Cl N O (M + H ), 345.1243; found, 345.1249.
15
23
2
4
6
.1.40. 3-(2,6-Dichloro-9H-purin-9-yl)undecan-1-ol (8c). 2,6-
Dichloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
4
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
compound 5d as a white solid in 47.3 mg (85.1% yield). White solid.
2
4
1
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 8c as a white solid in 56.4 mg (78.8% yield). White solid.
mp: 112−113 °C. H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H), 4.81−
mp: 73−75 °C. H NMR (400 MHz, CDCl ) δ 8.48 (s, 1H), 7.92 (s,
3
1H), 4.81−4.72 (m, 1H), 4.16 (s, 3H), 3.61−3.49 (m, 2H), 3.18−
3.08 (m, 1H), 2.21−2.08 (m, 2H), 2.02−1.88 (m, 2H), 1.29−1.18
1
3
1
3
4
2
1
.70 (m, 1H), 3.68−3.59 (m, 1H), 3.36−3.26 (m, 1H), 2.42 (s, 1H),
(m, 5H), 1.15−1.07 (m, 1H), 0.79 (t, J = 7.2 Hz, 3H); C NMR
.24−2.16 (m, 2H), 2.15−2.06 (m, 1H), 2.00−1.88 (m, 1H), 1.29−
(100 MHz, CDCl ) δ 161.3, 152.4, 152.0, 140.9, 121.5, 58.1, 54.4,
3
1
3
.14 (m, 11H), 1.11−1.01 (m, 1H), 0.82 (t, J = 6.8 Hz, 3H);
C
52.9, 38.4, 34.5, 31.3, 26.0, 22.4, 14.0; ESI-HRMS (m/z) calcd
+
NMR (100 MHz, CDCl ) δ 153.3, 152.7, 151.8, 145.5, 131.0, 58.5,
C H N O (M + H ), 279.1816; found, 279.1812.
3
14 23
4
2
5
4.9, 37.0, 34.3, 31.8, 29.3, 29.2, 29.1, 26.3, 22.7, 14.1; ESI-HRMS
6.1.44. 3-(6-Methoxy-9H-purin-9-yl)nonan-1-ol (6d). 6-Methoxy-
9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-non-
2-enal (49.7 μL, 0.3 mmol) was added to the reaction mixture. The
reaction was stirred at room temperature for 24 h and detected by
+
(m/z) calcd C H Cl N O (M + H ), 359.1400; found, 359.1399.
16 25 2 4
6
.1.41. 3-(2,6-Dichloro-9H-purin-9-yl)dodecan-1-ol (9c). 2,6-
Dichloro-9H-purine (37.8 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
4
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
times. The combined organic extracts were dried over Na SO and
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
compound 6d as a white solid in 48.8 mg (83.6% yield). White solid.
2
4
1
column chromatography on silica gel (PE/EA = 1:1) to give the target
compound 9c as a white solid in 62.6 mg (84.1% yield). White solid.
mp: 73−74 °C. H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H), 7.93 (s,
3
1H), 4.82−4.73 (m, 1H), 4.18 (s, 3H), 3.62−3.52 (m, 1H), 3.40 (s,
1H), 3.18−3.07 (m, 1H), 2.24−2.06 (m, 2H), 2.00−1.92 (m, 2H),
1.30−1.14 (m, 8H), 0.81 (t, J = 7.2 Hz, 3H); C NMR (100 MHz,
1
mp: 94−96 °C. H NMR (600 MHz, CDCl ) δ 8.15 (s, 1H), 4.78−
3
1
3
4.71 (m, 1H), 3.66−3.60 (m, 1H), 3.34−3.28 (m, 1H), 2.74 (s, 1H),
2
096
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
CDCl ) δ 161.3, 152.5, 152.0, 140.8, 121.5, 58.1, 54.4, 52.8, 38.6,
6.1.48. 3-(6-Methoxy-9H-purin-9-yl)tridecan-1-ol (10d). 6-Me-
thoxy-9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-
tridec-2-enal (58.9 mg, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
(22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
3
3
4.5, 31.6, 28.8, 26.3, 22.5, 14.1; ESI-HRMS (m/z) calcd
+
C H N O (M + H ), 293.1972; found, 293.1973.
15
25
4
2
6
.1.45. 3-(6-Methoxy-9H-purin-9-yl)decan-1-ol (7d). 6-Methoxy-
9
H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol),
benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were
added to a reaction tube with a magnetic stirring bar. Then, (E)-dec-
2
-enal (54.7 μL, 0.3 mmol) was added to the reaction mixture. The
reaction was stirred at room temperature for 24 h and detected by
TLC. Then, the solution was cooled to 0 °C. After that, NaBH (22.7
4
mg, 0.6 mmol) was slowly added and the reaction was stirred for 10
min. Then, the reaction was quenched with a saturated ammonium
chloride solution and concentrated in vacuo. Then, water was added
to the residue and the mixture was extracted with ethyl acetate three
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
times. The combined organic extracts were dried over Na SO and
2
4
compound 10d as a white solid in 57.1 mg (82.1% yield). White solid.
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
1
mp: 49−51 °C. H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H), 7.93 (s,
3
1
H), 4.83−4.73 (m, 1H), 4.18 (s, 3H), 3.62−3.53 (m, 1H), 3.39 (s,
H), 3.13 (td, J = 11.2, 3.6 Hz, 1H), 2.24−2.07 (m, 2H), 2.01−1.90
compound 7d as a white solid in 53.0 mg (86.7% yield). White solid.
1
1
mp: 69−70 °C. H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H), 7.93 (s,
3
(m, 2H), 1.28−1.22 (m, 5H), 1.22−1.16 (m, 11H), 0.85 (t, J = 6.8
1
1
1
H), 4.83−4.73 (m, 1H), 4.18 (s, 3H), 3.64−3.50 (m, 1H), 3.40 (s,
13
Hz, 3H); C NMR (150 MHz, CDCl ) δ 161.3, 152.5, 152.0, 140.8,
3
H), 3.18−3.06 (m, 1H), 2.23−2.06 (m, 2H), 2.00−1.93 (m, 2H),
1
21.5, 58.1, 54.4, 52.8, 38.6, 34.5, 32.0, 29.6, 29.5, 29.42, 29.36, 29.2,
1
3
.29−1.13 (m, 10H), 0.82 (t, J = 6.8 Hz, 3H); C NMR (100 MHz,
+
26.4, 22.8, 14.2; ESI-HRMS (m/z) calcd C H N O (M + H ),
1
9
33
4
2
CDCl ) δ 161.3, 152.5, 152.0, 140.8, 121.5, 58.1, 54.4, 52.8, 38.6,
3
349.2598; found, 349.2606.
3
4.5, 31.7, 29.12, 29.08, 26.4, 22.6, 14.1; ESI-HRMS (m/z) calcd
6
.1.49. 3-(6-Methoxy-9H-purin-9-yl)tetradecan-1-ol (11d). 6-
+
C H N O (M + H ), 307.2129; found, 307.2130.
16
27
4
2
Methoxy-9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
tetradec-2-enal (63.1 mg, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
6
.1.46. 3-(6-Methoxy-9H-purin-9-yl)undecan-1-ol (8d). 6-Me-
thoxy-9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
2
4
compound 11d as a white solid in 61.9 mg (85.5% yield). White solid.
column chromatography on silica gel (PE/EA = 1:1) to give the target
1
mp: 57−58 °C. H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H), 7.93 (s,
3
compound 8d as a white solid in 57.3 mg (89.5% yield). White solid.
1
1H), 4.83−4.73 (m, 1H), 4.18 (s, 3H), 3.62−3.53 (m, 1H), 3.37 (s,
mp: 68−70 °C. H NMR (400 MHz, CDCl ) δ 8.48 (s, 1H), 7.92 (s,
3
1
H), 3.12 (td, J = 11.2, 3.6 Hz, 1H), 2.24−2.06 (m, 2H), 2.01−1.90
1
H), 4.82−4.72 (m, 1H), 4.16 (s, 3H), 3.69−3.29 (m, 2H), 3.13 (td,
(
m, 2H), 1.29−1.23 (m, 5H), 1.23−1.16 (m, 13H), 0.85 (t, J = 7.2
J = 10.8, 3.6 Hz, 1H), 2.22−2.06 (m, 2H), 2.02−1.88 (m, 2H), 1.26−
13
1
3
Hz, 3H); C NMR (100 MHz, CDCl ) δ 161.3, 152.5, 152.0, 140.8,
3
1
.20 (m, 4H), 1.20−1.10 (m, 8H), 0.82 (t, J = 7.2 Hz, 3H); C NMR
1
2
21.5, 58.1, 54.4, 52.8, 38.6, 34.5, 32.0, 29.66, 29.65, 29.5, 29.43,
(
100 MHz, CDCl ) δ 161.3, 152.4, 152.0, 140.9, 121.5, 58.1, 54.4,
3
9.41, 29.2, 26.4, 22.8, 14.2; ESI-HRMS (m/z) calcd C H N O (M
2
0
35
4
2
5
2.9, 38.5, 34.5, 31.8, 29.4, 29.2, 29.1, 26.4, 22.7, 14.1; ESI-HRMS
+
+
+ H ), 363.2755; found, 363.2759.
.1.50. 3-(6-(Methylamino)-9H-purin-9-yl)octan-1-ol (5e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-oct-2-enal (44.8 μL, 0.3 mmol) was added to the reaction
(m/z) calcd C H N O (M + H ), 321.2285; found, 321.2289.
17 29 4 2
6
6
.1.47. 3-(6-Methoxy-9H-purin-9-yl)dodecan-1-ol (9d). 6-Me-
thoxy-9H-purine (30.0 mg, 0.2 mmol), L-proline (2.3 mg, 0.02
mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL)
were added to a reaction tube with a magnetic stirring bar. Then, (E)-
dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction mixture.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, the solution was cooled to 0 °C. After that, NaBH4
0
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
(
22.7 mg, 0.6 mmol) was slowly added and the reaction was stirred
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
2
4
column chromatography on silica gel (PE/EA = 1:1) to give the target
column chromatography on silica gel (CH
Cl /MeOH = 25:1) to
2 2
compound 9d as a white solid in 58.9 mg (88.1% yield). White solid.
give the target compound 5e as a white solid in 16.1 mg (29.1%
1
1
mp: 66−68 °C. H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H), 7.93 (s,
yield). White solid. mp: 109−111 °C. H NMR (400 MHz, CDCl ) δ
3
3
1
H), 4.83−4.72 (m, 1H), 4.18 (s, 3H), 3.61−3.53 (m, 1H), 3.41 (s,
H), 3.13 (td, J = 10.8, 3.6 Hz, 1H), 2.23−2.06 (m, 2H), 2.00−1.92
8.33 (s, 1H), 7.72 (s, 1H), 6.48−6.41 (m, 1H), 4.75−4.65 (m, 1H),
4.34 (s, 1H), 3.57−3.50 (m, 1H), 3.23−3.08 (m, 4H), 2.19−2.09 (m,
1
(
m, 2H), 1.28−1.21 (m, 5H), 1.21−1.13 (m, 9H), 0.84 (t, J = 6.8 Hz,
1H), 2.09−2.00 (m, 1H), 1.95−1.83 (m, 2H), 1.25−1.10 (m, 6H),
H); 13C NMR (100 MHz, CDCl ) δ 161.3, 152.5, 152.0, 140.9,
13
3
0.79 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl
) δ 155.7,
3
3
1
21.5, 58.1, 54.4, 52.8, 38.6, 34.5, 31.9, 29.5, 29.4, 29.3, 29.2, 26.4,
153.1, 149.2, 138.0, 119.6, 57.9, 52.3, 38.8, 34.5, 31.3, 27.7, 26.1, 22.5,
+
+
22.7, 14.2; ESI-HRMS (m/z) calcd C H N O (M + H ), 335.2442;
14.0; ESI-HRMS (m/z) calcd C H N O (M + H ), 278.1975;
1
8
31
4
2
14 24
5
found, 335.2448.
found, 278.1985.
2
097
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J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
6
.1.51. 3-(6-(Methylamino)-9H-purin-9-yl)nonan-1-ol (6e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-non-2-enal (49.7 μL, 0.3 mmol) was added to the reaction
6.1.54. 3-(6-(Methylamino)-9H-purin-9-yl)dodecan-1-ol (9e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
0
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
2
4
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
2
2
2
2
give the target compound 9e as a white solid in 18.5 mg (27.8%
give the target compound 6e as a white solid in 15.5 mg (26.7%
1
1
yield). White solid. mp: 92−94 °C. H NMR (400 MHz, CDCl ) δ
yield). White solid. mp: 94−96 °C. H NMR (400 MHz, CDCl ) δ
3
3
8
.38 (s, 1H), 7.75 (s, 1H), 5.93 (s, 1H), 4.81−4.66 (m, 1H), 4.01 (s,
H), 3.61−3.51 (m, 1H), 3.22 (s, 3H), 3.09 (t, J = 11.6 Hz, 1H),
.25−2.13 (m, 1H), 2.13−2.04 (m, 1H), 2.00−1.89 (m, 1H), 1.81 (t,
8
1
2
.33 (s, 1H), 7.74 (s, 1H), 6.41 (s, 1H), 4.75−4.66 (m, 1H), 4.31 (s,
H), 3.59−3.51 (m, 1H), 3.26−3.06 (m, 4H), 2.20−2.10 (m, 1H),
.10−2.00 (m, 1H), 1.97−1.83 (m, 2H), 1.27−1.12 (m, 8H), 0.80 (t,
1
2
1
3
13
J = 12.8 Hz, 1H), 1.29−1.18 (m, 14H), 0.86 (t, J = 7.2 Hz, 3H). C
J = 6.8 Hz, 3H); C NMR (150 MHz, CDCl ) δ 155.7, 153.1, 149.3,
3
NMR (100 MHz, CDCl ) δ 155.7, 153.1, 149.4, 137.9, 119.6, 57.9,
1
37.9, 119.7, 57.9, 52.2, 39.0, 34.5, 31.6, 28.9, 27.6, 26.4, 22.6, 14.1;
3
+
52.3, 38.9, 34.5, 31.9, 29.5, 29.4, 29.3, 29.2, 27.7, 26.4, 22.7, 14.2; ESI-
ESI-HRMS (m/z) calcd C H N O (M + H ), 292.2132; found,
1
5
26
5
+
HRMS (m/z) calcd C H N O (M + H ), 334.2601; found,
2
92.2137.
.1.52. 3-(6-(Methylamino)-9H-purin-9-yl)decan-1-ol (7e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-dec-2-enal (54.7 μL, 0.3 mmol) was added to the reaction
18 32
5
3
34.2600.
.1.55. 3-(6-(Methylamino)-9H-purin-9-yl)tridecan-1-ol (10e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-tridec-2-enal (58.9 mg, 0.3 mmol) was added to the reaction
6
6
0
0
(
(
mixture. The reaction was stirred at room temperature for 24 h and
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
detected by TLC. Then, the solution was cooled to 0 °C. After that,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
2
4
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
2
2
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
2
2
give the target compound 7e as a white solid in 19.0 mg (31.1%
give the target compound 10e as a white solid in 19.3 mg (27.8%
1
yield). White solid. mp: 68−69 °C. H NMR (400 MHz, CDCl ) δ
1
3
yield). White solid. mp: 76−77 °C. H NMR (400 MHz, CDCl ) δ
3
8
4
1
0
1
2
3
.34 (s, 1H), 7.73 (s, 1H), 6.39−6.30 (m, 1H), 4.76−4.65 (m, 1H),
.32 (s, 1H), 3.58−3.50 (m, 1H), 3.23−3.06 (m, 4H), 2.20−2.10 (m,
H), 2.10−2.01 (m, 1H), 1.97−1.82 (m, 2H), 1.27−1.13 (m, 10H),
8
1
2
.37 (s, 1H), 7.74 (s, 1H), 6.09 (s, 1H), 4.78−4.65 (m, 1H), 4.08 (s,
H), 3.60−3.51 (m, 1H), 3.21 (s, 3H), 3.14−3.04 (m, 1H), 2.22−
.13 (m, 1H), 2.13−2.04 (m, 1H), 2.00−1.89 (m, 1H), 1.87−1.76
1
3
.81 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 155.6,
3
(
m, 1H), 1.29−1.24 (m, 6H), 1.23−1.19 (m, 10H), 0.86 (t, J = 6.8
53.1, 149.2, 138.0, 119.6, 57.9, 52.3, 38.8, 34.5, 31.7, 29.12, 29.06,
13
Hz, 3H); C NMR (150 MHz, CDCl ) δ 155.7, 153.2, 149.4, 137.8,
+
3
7.6, 26.4, 22.6, 14.1; ESI-HRMS (m/z) calcd C H N O (M + H ),
1
6
28
5
1
2
3
19.7, 58.0, 52.0, 39.2, 34.5, 32.0, 29.6, 29.6, 29.5, 29.4, 29.2, 27.7,
06.2288; found, 306.2288.
.1.53. 3-(6-(Methylamino)-9H-purin-9-yl)undecan-1-ol (8e). N-
Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction
+
6.5, 22.8, 14.2; ESI-HRMS (m/z) calcd C H N O (M + H ),
1
9
34
5
6
48.2758; found, 348.2753.
.1.56. 3-(6-(Methylamino)-9H-purin-9-yl)tetradecan-1-ol (11e).
N-Methyl-9H-purin-6-amine (29.8 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-tetradec-2-enal (63.1 mg, 0.3 mmol) was added to the reaction
6
0
0
(
mixture. The reaction was stirred at room temperature for 24 h and
(
detected by TLC. Then, the solution was cooled to 0 °C. After that,
mixture. The reaction was stirred at room temperature for 24 h and
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
detected by TLC. Then, the solution was cooled to 0 °C. After that,
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
2
2
2
4
give the target compound 8e as a white solid in 20.9 mg (32.8%
column chromatography on silica gel (CH Cl /MeOH = 25:1) to
2 2
1
yield). White solid. mp: 88−90 °C. H NMR (400 MHz, CDCl ) δ
give the target compound 11e as a white solid in 21.5 mg (29.8%
3
1
8
1
2
1
.35 (s, 1H), 7.77 (s, 1H), 6.24 (s, 1H), 4.78−4.67 (m, 1H), 3.96 (s,
H), 3.61−3.52 (m, 1H), 3.32−3.05 (m, 4H), 2.23−2.11 (m, 1H),
.10−2.02 (m, 1H), 1.99−1.82 (m, 2H), 1.30−1.22 (m, 5H), 1.22−
yield). White solid. mp: 90−91 °C. H NMR (400 MHz, CDCl ) δ
3
8.37 (s, 1H), 7.75 (s, 1H), 6.03 (s, 1H), 4.79−4.68 (m, 1H), 4.04 (s,
1H), 3.60−3.52 (m, 1H), 3.21 (s, 3H), 3.13−3.05 (m, 1H), 2.23−
2.12 (m, 1H), 2.12−2.04 (m, 1H), 1.99−1.90 (m, 1H), 1.85−1.77
(m, 1H), 1.30−1.23 (m, 8H), 1.23−1.18 (m, 10H), 0.86 (t, J = 6.8
1
3
.17 (m, 5H), 0.84 (t, J = 6.8 Hz, 3H). C NMR (150 MHz, CDCl )
3
δ 155.7, 153.1, 149.2, 138.0, 119.7, 57.9, 52.3, 38.9, 34.5, 31.8, 29.4,
1
3
2
9.2, 27.6, 26.4, 22.7, 14.1; ESI-HRMS (m/z) calcd C H N O (M +
Hz, 3H); C NMR (150 MHz, CDCl ) δ 155.7, 153.2, 149.3, 137.8,
1
7
30
5
3
+
H ), 320.2445; found, 320.2440.
119.7, 58.0, 52.0, 39.3, 34.5, 32.0, 29.8, 29.6, 29.6, 29.5, 29.4, 29.3,
2
098
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
2
3
7.7, 26.5, 22.8, 14.2; ESI-HRMS (m/z) calcd C H N O (M + H ),
6.1.60. 3-(6-(Dimethylamino)-9H-purin-9-yl)undecan-1-ol (8f).
N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline
(2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-undec-2-enal (59.5 μL, 0.3 mmol) was added
to the reaction mixture. The reaction was stirred at room temperature
for 24 h and detected by TLC. Then, the solution was cooled to 0 °C.
2
0
36
5
62.2914; found, 362.2919.
.1.57. 3-(6-(Dimethylamino)-9H-purin-9-yl)octan-1-ol (5f). N,N-
Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-oct-2-enal (44.8 μL, 0.3 mmol) was added to the reaction
6
0
(
mixture. The reaction was stirred at room temperature for 24 h and
After that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the
4
detected by TLC. Then, the solution was cooled to 0 °C. After that,
reaction was stirred for 10 min. Then, the reaction was quenched with
a saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
purified by flash column chromatography on silica gel (PE/EA = 1:1)
2
4
to give the target compound 8f as a colorless oil in 52.7 mg (79.2%
1
column chromatography on silica gel (PE/EA = 1:1) to give the target
yield). Colorless oil. H NMR (400 MHz, CDCl ) δ 8.24 (s, 1H),
3
compound 5f as a colorless oil in 45.6 mg (78.3% yield). Colorless oil.
7.70 (s, 1H), 4.74−4.63 (m, 1H), 4.03 (s, 1H), 3.73−3.18 (m, 7H),
3.05 (td, J = 10.8, 3.2 Hz, 1H), 2.16−1.96 (m, 2H), 1.93−1.82 (m,
1H), 1.82−1.71 (m, 1H), 1.25−1.09 (m, 12H), 0.79 (t, J = 6.8 Hz,
1
H NMR (400 MHz, CDCl ) δ 8.24 (s, 1H), 7.71 (s, 1H), 4.74−4.64
3
(
m, 1H), 3.96 (s, 1H), 3.72−3.30 (m, 7H), 3.05 (td, J = 11.2, 3.2 Hz,
1
3
1
1
H), 2.18−1.96 (m, 2H), 1.94−1.83 (m, 1H), 1.82−1.70 (m, 1H),
3H); C NMR (100 MHz, CDCl ) δ 155.1, 152.3, 151.0, 136.2,
3
1
3
.30−1.13 (m, 6H), 0.78 (t, J = 6.8 Hz, 3H); C NMR (100 MHz,
119.8, 57.8, 51.5, 39.3, 38.6, 34.5, 31.8, 29.4, 29.18, 29.17, 26.4, 22.7,
+
CDCl ) δ 155.1, 152.3, 151.0, 136.2, 119.8, 57.8, 51.5, 39.3, 38.7,
14.1; ESI-HRMS (m/z) calcd C H N O (M + H ), 334.2601;
3
18 32
5
3
4.4, 31.4, 26.1, 22.5, 14.0; ESI-HRMS (m/z) calcd C H N O (M +
found, 334.2603.
1
5
26
5
+
H ), 292.2132; found, 292.2131.
.1.58. 3-(6-(Dimethylamino)-9H-purin-9-yl)nonan-1-ol (6f).
N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline
2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
6.1.61. 3-(6-(Dimethylamino)-9H-purin-9-yl)dodecan-1-ol (9f).
N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline
(2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-dodec-2-enal (64.4 μL, 0.3 mmol) was added
to the reaction mixture. The reaction was stirred at room temperature
for 24 h and detected by TLC. Then, the solution was cooled to 0 °C.
6
(
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-non-2-enal (49.7 μL, 0.3 mmol) was added to
the reaction mixture. The reaction was stirred at room temperature
for 24 h and detected by TLC. Then, the solution was cooled to 0 °C.
After that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the
4
After that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the
reaction was stirred for 10 min. Then, the reaction was quenched with
a saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
4
reaction was stirred for 10 min. Then, the reaction was quenched with
a saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
2
4
purified by flash column chromatography on silica gel (PE/EA = 1:1)
2
4
purified by flash column chromatography on silica gel (PE/EA = 1:1)
to give the target compound 9f as a colorless oil in 51.6 mg (74.3%
1
to give the target compound 6f as a colorless oil in 47.0 mg (77.1%
yield). Colorless oil. H NMR (400 MHz, CDCl ) δ 8.20 (s, 1H),
3
1
yield). Colorless oil. H NMR (400 MHz, CDCl ) δ 8.24 (s, 1H),
7.68 (s, 1H), 4.72−4.60 (m, 1H), 4.19 (s, 1H), 3.71−3.19 (m, 7H),
3
7
3
.70 (s, 1H), 4.73−4.64 (m, 1H), 3.94 (s, 1H), 3.68−3.27 (m, 7H),
.05 (td, J = 10.8, 3.2 Hz, 1H), 2.17−2.06 (m, 1H), 2.05−1.96 (m,
H), 1.94−1.83 (m, 1H), 1.82−1.72 (m, 1H), 1.28−1.08 (m, 8H),
3.05 (td, J = 11.2, 3.6 Hz, 1H), 2.14−1.92 (m, 2H), 1.92−1.69 (m,
13
2H), 1.25−1.03 (m, 14H), 0.77 (t, J = 6.8 Hz, 3H); C NMR (100
1
MHz, CDCl ) δ 155.0, 152.2, 150.9, 136.3, 119.8, 57.8, 51.6, 39.2,
3
1
3
0.78 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 155.0,
38.6, 34.5, 31.9, 29.42, 29.37, 29.2, 29.1, 26.3, 22.7, 14.1; ESI-HRMS
3
+
52.3, 151.0, 136.3, 119.8, 57.8, 51.6, 39.3, 38.7, 34.5, 31.6, 28.9, 26.3,
(m/z) calcd C H N O (M + H ), 348.2758; found, 348.2764.
1
9
34
5
+
1
6
28
5
6.1.62. 3-(6-(Dimethylamino)-9H-purin-9-yl)tridecan-1-ol (10f).
N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline (2.3
mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol
(1.5 mL) were added to a reaction tube with a magnetic stirring bar.
Then, (E)-tridec-2-enal (58.9 mg, 0.3 mmol) was added to the
reaction mixture. The reaction was stirred at room temperature for 24
h and detected by TLC. Then, the solution was cooled to 0 °C. After
found, 306.2295.
6
.1.59. 3-(6-(Dimethylamino)-9H-purin-9-yl)decan-1-ol (7f).
N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-proline
2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
(
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-dec-2-enal (54.7 μL, 0.3 mmol) was added to
the reaction mixture. The reaction was stirred at room temperature
for 24 h and detected by TLC. Then, the solution was cooled to 0 °C.
that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction
4
was stirred for 10 min. Then, the reaction was quenched with a
saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
After that, NaBH (22.7 mg, 0.6 mmol) was slowly added and the
4
reaction was stirred for 10 min. Then, the reaction was quenched with
a saturated ammonium chloride solution and concentrated in vacuo.
Then, water was added to the residue and the mixture was extracted
with ethyl acetate three times. The combined organic extracts were
dried over Na SO and concentrated in vacuo. The residue was
2
4
purified by flash column chromatography on silica gel (PE/EA = 1:1)
2
4
to give the target compound 10f as a colorless oil in 55.3 mg (76.6%
1
purified by flash column chromatography on silica gel (PE/EA = 1:1)
yield). Colorless oil. H NMR (400 MHz, CDCl ) δ 8.29 (s, 1H),
3
to give the target compound 7f as a colorless oil in 51.1 mg (80.1%
7.73 (s, 1H), 4.78−4.68 (m, 1H), 4.27 (s, 1H), 3.75−3.33 (m, 7H),
3.09−2.99 (m, 1H), 2.22−2.10 (m, 1H), 2.10−2.01 (m, 1H), 1.99−
1.87 (m, 2H), 1.79−1.67 (m, 1H), 1.28−1.23 (m, 6H), 1.22−1.18
1
yield). Colorless oil. H NMR (400 MHz, CDCl ) δ 8.24 (s, 1H),
3
7
3
1
3
1
.70 (s, 1H), 4.74−4.64 (m, 1H), 4.09 (s, 1H), 3.75−3.23 (m, 7H),
.05 (td, J = 11.2, 3.2 Hz, 1H), 2.16−1.96 (m, 2H), 1.94−1.82 (m,
H), 1.82−1.71 (m, 1H), 1.26−1.08 (m, 10H), 0.78 (t, J = 6.8 Hz,
1
3
(m, 10H), 0.85 (t, J = 6.8 Hz, 3H); C NMR (150 MHz, CDCl ) δ
3
155.1, 152.4, 151.1, 136.1, 119.9, 57.9, 51.4, 39.5, 38.7, 34.5, 32.0,
H); 13C NMR (100 MHz, CDCl ) δ 155.0, 152.3, 151.0, 136.3,
3
29.63, 29.57, 29.5, 29.4, 29.3, 26.5, 22.8, 14.2; ESI-HRMS (m/z)
+
19.8, 57.8, 51.6, 39.3, 38.7, 34.5, 31.7, 29.14, 29.06, 26.4, 22.6, 14.1;
calcd C H N O (M + H ), 362.2914; found, 362.2917.
2
0
36
5
+
ESI-HRMS (m/z) calcd C H N O (M + H ), 320.2445; found,
6.1.63. 3-(6-(Dimethylamino)-9H-purin-9-yl)tetradecan-1-ol
(11f). N,N-Dimethyl-9H-purin-6-amine (32.6 mg, 0.2 mmol), L-
1
7
30
5
320.2447.
2
099
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
proline (2.3 mg, 0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and
methanol (1.5 mL) were added to a reaction tube with a magnetic
stirring bar. Then, (E)-tetradec-2-enal (63.1 mg, 0.3 mmol) was
added to the reaction mixture. The reaction was stirred at room
temperature for 24 h and detected by TLC. Then, the solution was
target compound 7g as a white solid in 21.0 mg (72.3% yield). White
1
solid. mp: 124−126 °C. H NMR (400 MHz, CDCl ) δ 8.28 (s, 1H),
3
7.83 (s, 1H), 6.57 (s, 2H), 4.80−4.67 (m, 1H), 4.52 (s, 1H), 3.61−
3.54 (m, 1H), 3.22−3.14 (m, 1H), 2.22−2.12 (m, 1H), 2.11−2.03
(m, 1H), 2.01−1.86 (m, 2H), 1.27−1.12 (m, 10H), 0.81 (t, J = 6.4
1
3
cooled to 0 °C. After that, NaBH (22.7 mg, 0.6 mmol) was slowly
Hz, 3H); C NMR (100 MHz, CDCl ) δ 155.9, 152.9, 150.5, 138.9,
4
3
added and the reaction was stirred for 10 min. Then, the reaction was
quenched with a saturated ammonium chloride solution and
concentrated in vacuo. Then, water was added to the residue and
the mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na SO and concentrated
119.4, 58.0, 52.5, 38.8, 34.6, 31.8, 29.2, 29.1, 26.4, 22.7, 14.1; ESI-
+
HRMS (m/z) calcd C H N O (M + H ), 292.2132; found,
1
5
26
5
292.2132.
6.1.68. 3-(6-Amino-9H-purin-9-yl)undecan-1-ol (8g). Compound
8b (32.5 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH)
were added to a reaction tube with a magnetic stirring bar. The
reaction was stirred at 100 °C for 6 h and detected by TLC. Then, the
residue was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (CH Cl /MeOH = 20:1) to
2
4
in vacuo. The residue was purified by flash column chromatography
on silica gel (PE/EA = 1:1) to give the target compound 11f as a
1
colorless oil in 55.0 mg (73.3% yield). Colorless oil. H NMR (400
MHz, CDCl ) δ 8.28 (s, 1H), 7.73 (s, 1H), 4.78−4.67 (m, 1H), 4.29
3
2
2
(
s, 1H), 3.88−3.26 (m, 7H), 3.11−2.99 (m, 1H), 2.22−2.10 (m, 1H),
give the target compound 8g as a white solid in 21.1 mg (69.1%
2
.09−2.00 (m, 1H), 2.00−1.87 (m, 1H), 1.29−1.17 (m, 18H), 0.85
1
yield). White solid. mp: 136−138 °C. H NMR (400 MHz, CDCl ) δ
3
1
3
(
t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 155.1, 152.3,
3
8.26 (s, 1H), 7.81 (s, 1H), 6.69 (s, 2H), 4.76−4.66 (m, 1H), 4.60 (s,
1
2
51.1, 136.2, 119.8, 57.8, 51.5, 39.4, 38.7, 34.5, 32.0, 29.67, 29.66,
1
H), 3.60−3.53 (m, 1H), 3.23−3.14 (m, 1H), 2.20−2.10 (m, 1H),
9.6, 29.5, 29.4, 29.2, 26.4, 22.8, 14.2; ESI-HRMS (m/z) calcd
2
.10−2.02 (m, 1H), 2.01−1.85 (m, 2H), 1.27−1.20 (m, 4H), 1.20−
+
C H N O (M + H ), 376.3071; found, 376.3076.
13
21
38
5
1.10 (m, 8H), 0.81 (t, J = 6.4 Hz, 3H); C NMR (100 MHz, CDCl )
3
6
.1.64. 3-(6-Amino-9H-purin-9-yl)heptan-1-ol (4g). Compound
δ 155.9, 152.9, 150.4, 138.9, 119.4, 58.0, 52.5, 38.7, 34.6, 31.8, 29.4,
4
b (26.9 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH)
+
2
9.2, 26.4, 22.7, 14.2; ESI-HRMS (m/z) calcd C H N O (M + H ),
1
6
28
5
were added to a reaction tube with a magnetic stirring bar. The
reaction was stirred at 100 °C for 6 h and detected by TLC. Then, the
residue was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (CH Cl /MeOH = 20:1) to
3
06.2288; found, 306.2287.
.1.69. 3-(6-Amino-9H-purin-9-yl)dodecan-1-ol (9g). Compound
b (33.9 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH)
6
9
2
2
were added to a reaction tube with a magnetic stirring bar. The
reaction was stirred at 100 °C for 6 h and detected by TLC. Then, the
residue was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (CH Cl /MeOH = 20:1) to
give the target compound 4g as a white solid in 17.2 mg (69.1%
1
yield). White solid. mp: 118−121 °C. H NMR (400 MHz, CDCl ) δ
3
8
1
2
.30 (s, 1H), 7.81 (s, 1H), 6.27 (s, 2H), 4.79−4.67 (m, 1H), 4.19 (s,
H), 3.61−3.53 (m, 1H), 3.20−3.09 (m, 1H), 2.23−2.03 (m, 2H),
.00−1.86 (m, 2H), 1.34−1.22 (m, 3H), 1.21−1.09 (m, 1H), 0.83 (t,
2
2
give the target compound 9g as a white solid in 22.2 mg (69.5%
1
yield). White solid. mp: 120−122 °C. H NMR (400 MHz, CDCl ) δ
13
3
J = 6.8 Hz, 3H); C NMR (150 MHz, CDCl ) δ 155.9, 152.9, 150.5,
3
8
1
2
1
.28 (s, 1H), 7.82 (s, 1H), 6.61 (s, 2H), 4.79−4.66 (m, 1H), 4.51 (s,
H), 3.62−3.52 (m, 1H), 3.23−3.13 (m, 1H), 2.22−2.11 (m, 1H),
.11−2.02 (m, 1H), 2.01−1.85 (m, 2H), 1.31−1.21 (m, 5H), 1.20−
1
38.9, 119.5, 58.0, 52.4, 38.8, 34.2, 28.6, 22.3, 14.0; ESI-HRMS (m/z)
+
calcd C H N O (M + H ), 250.1662; found, 250.1664.
12
20
5
6
.1.65. 3-(6-Amino-9H-purin-9-yl)octan-1-ol (5g). Compound 5b
1
3
.14 (m, 9H), 0.83 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl )
3
(
28.3 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH) were
δ 155.9, 152.9, 150.3, 138.9, 119.4, 58.0, 52.6, 38.6, 34.6, 31.9, 29.5,
added to a reaction tube with a magnetic stirring bar. The reaction
was stirred at 100 °C for 6 h and detected by TLC. Then, the residue
was concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH Cl /MeOH = 20:1) to give the
2
9.4, 29.3, 29.2, 26.4, 22.7, 14.2; ESI-HRMS (m/z) calcd C H N O
1
7
30
5
+
(M + H ), 320.2445; found, 320.2455.
6
.1.70. 3-(6-Amino-9H-purin-9-yl)tridecan-1-ol (10g). Com-
2
2
pound 10b (35.3 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in
MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
target compound 5g as a white solid in 19.6 mg (74.1% yield). White
1
solid. mp: 109−111 °C. H NMR (400 MHz, CDCl ) δ 8.29 (s, 1H),
3
7
.83 (s, 1H), 6.55 (s, 2H), 4.79−4.67 (m, 1H), 4.50 (s, 1H), 3.61−
.54 (m, 1H), 3.23−3.13 (m, 1H), 2.22−2.12 (m, 1H), 2.11−2.03
3
(
2
2
m, 1H), 2.00−1.88 (m, 2H), 1.28−1.14 (m, 6H), 0.80 (t, J = 6.8 Hz,
H); 13C NMR (100 MHz, CDCl ) δ 155.8, 153.0, 150.6, 138.8,
20:1) to give the target compound 10g as a white solid in 22.6 mg
3
1
3
1
(
67.9% yield). White solid. mp: 105−106 °C. H NMR (400 MHz,
19.5, 58.0, 52.3, 39.0, 34.5, 31.4, 26.1, 22.5, 14.0; ESI-HRMS (m/z)
calcd C H N O (M + H ), 264.1819; found, 264.1818.
+
CDCl ) δ 8.31 (s, 1H), 7.81 (s, 1H), 6.11 (s, 2H), 4.79−4.69 (m,
3
13
22
5
1
2
(
(
H), 4.06 (s, 1H), 3.61−3.54 (m, 1H), 3.19−3.09 (m, 1H), 2.23−
.13 (m, 1H), 2.13−2.04 (m, 1H), 2.00−1.83 (m, 2H), 1.28−1.23
6
.1.66. 3-(6-Amino-9H-purin-9-yl)nonan-1-ol (6g). Compound
6
b (29.7 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH)
1
3
m, 5H), 1.23−1.18 (m, 11H), 0.85 (t, J = 6.8 Hz, 3H); C NMR
were added to a reaction tube with a magnetic stirring bar. The
reaction was stirred at 100 °C for 6 h and detected by TLC. Then, the
residue was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (CH Cl /MeOH = 20:1) to
100 MHz, CDCl ) δ 155.8, 153,0, 150.5, 138.9, 119.4, 58.0, 52.4,
3
3
8.9, 34.5, 32.0, 29.63, 29.58, 29.5, 29.4, 29.2, 26.5, 22.8, 14.2; ESI-
+
HRMS (m/z) calcd C H N O (M + H ), 334.2601; found,
18 32
5
2
2
3
34.2605.
give the target compound 6g as a white solid in 21.0 mg (75.9%
1
6
.1.71. 3-(6-Amino-9H-purin-9-yl)tetradecan-1-ol (11g). Com-
yield). White solid. mp: 119−120 °C. H NMR (400 MHz, CDCl ) δ
3
pound 11b (36.7 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in
MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
8
1
2
.27 (s, 1H), 7.82 (s, 1H), 6.62 (s, 2H), 4.77−4.68 (m, 1H), 4.63 (s,
H), 3.61−3.53 (m, 1H), 3.23−3.14 (m, 1H), 2.22−2.11 (m, 1H),
.11−2.02 (m, 1H), 2.02−1.86 (m, 2H), 1.28−1.13 (m, 8H), 0.80 (t,
13
J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 155.8, 153.0, 150.5,
3
1
38.9, 119.5, 58.0, 52.4, 38.9, 34.5, 31.6, 28.9, 26.4, 22.6, 14.1; ESI-
by flash column chromatography on silica gel (CH Cl /MeOH =
2 2
+
HRMS (m/z) calcd C H N O (M + H ), 278.1975; found,
20:1) to give the target compound 11g as a white solid in 24.7 mg
1
4
24
5
1
2
78.1981.
.1.67. 3-(6-Amino-9H-purin-9-yl)decan-1-ol (7g). Compound 7b
31.1 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in MeOH) were
(71.1% yield). White solid. mp: 93−94 °C. H NMR (400 MHz,
6
CDCl ) δ 8.31 (s, 1H), 7.81 (s, 1H), 6.12 (s, 2H), 4.78−4.69 (m,
3
(
1H), 4.05 (s, 1H), 3.61−3.54 (m, 1H), 3.18−3.09 (m, 1H), 2.24−
2.12 (m, 1H), 2.12−2.04 (m, 1H), 2.01−1.83 (m, 2H), 1.28−1.24
added to a reaction tube with a magnetic stirring bar. The reaction
was stirred at 100 °C for 6 h and detected by TLC. Then, the residue
was concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH Cl /MeOH = 20:1) to give the
1
3
(m, 5H), 1.24−1.18 (m, 13H), 0.86 (t, J = 6.8 Hz, 3H); C NMR
(100 MHz, CDCl ) δ 155.8, 153.0, 150.5, 138.9, 119.4, 58.0, 52.4,
3
38.9, 34.5, 32.0, 29.7, 29.6, 29.5, 29.4, 29.2, 26.5, 22.8, 14.2; ESI-
2
2
2
100
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

Journal of Medicinal Chemistry
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Article
+
HRMS (m/z) calcd C H N O (M + H ), 348.2758; found,
CDCl ) δ 7.89 (s, 1H), 6.82 (s, 2H), 4.73−4.62 (m, 1H), 3.80 (s,
1
9
34
5
3
3
48.2768.
6
1H), 3.67−3.55 (m, 1H), 3.35−3.24 (m, 1H), 2.22−2.11 (m, 1H),
2.10−2.00 (m, 2H), 1.96−1.85 (m, 1H), 1.29−1.23 (m, 4H), 1.22−
.1.72. 3-(6-Amino-2-chloro-9H-purin-9-yl)octan-1-ol (5h). Com-
1
3
pound 5c (31.7 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M in
MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
1.17 (m, 9H), 1.14−1.08 (m, 1H), 0.84 (t, J = 7.2 Hz, 3H). C NMR
(150 MHz, CDCl ) δ 156.4, 154.1, 151.5, 139.5, 118.3, 58.4, 53.1,
3
38.3, 34.6, 32.0, 29.53, 29.47, 29.3, 29.2, 26.4, 22.8, 14.2; ESI-HRMS
+
(m/z) calcd C H ClN O (M + H ), 354.2055; found, 354.2054.
1
7
29
5
6.1.77. 3-(6-Amino-2-chloro-9H-purin-9-yl)tridecan-1-ol (10h).
3-(2,6-Dichloro-9H- purin-9-yl)tridecan-1-ol (38.7 mg, 0.1 mmol)
(for 3-(2,6-dichloro-9H-purin-9-yl)tridecan-1-ol: prepared according
to the procedure for the synthesis of 9c, using 2,6-dichloro-9H-purine
to react with (E)-tridec-2-enal) and ammonia (2.0 mL, 7.0 M in
MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
2
2
3
(
0:1) to give the target compound 5h as a white solid in 20.3 mg
1
68.3% yield). White solid. mp: 154−156 °C. H NMR (400 MHz,
CDCl ) δ 7.78 (s, 1H), 6.09 (s, 2H), 4.73−4.61 (m, 1H), 3.65−3.55
3
(
1
1
m, 1H), 3.29−3.18 (m, 1H), 2.98−2.86 (m, 1H), 2.24−2.12 (m,
H), 2.12−2.04 (m, 1H), 2.03−1.88 (m, 2H), 1.28−1.25 (m, 5H),
13
.20−1.14 (m, 1H), 0.84 (t, J = 6.8 Hz, 3H); C NMR (150 MHz,
CDCl ) δ 156.3, 154.1, 151.6, 139.5, 118.3, 58.4, 53.1, 38.4, 34.5,
3
3
1.4, 26.1, 22.5, 14.0; ESI-HRMS (m/z) calcd C H ClN O (M +
H ), 298.1429; found, 298.1433.
.1.73. 3-(6-Amino-2-chloro-9H-purin-9-yl)nonan-1-ol (6h).
Compound 6c (33.1 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M
in MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
13 21 5
2
2
+
30:1) to give the target compound 10h as a white solid in 25.5 mg
1
6
(69.5% yield). White solid. mp: 131−132 °C. H NMR (400 MHz,
CDCl ) δ 7.78 (s, 1H), 6.81 (s, 2H), 4.71−4.62 (m, 1H), 3.66−3.56
3
(m, 2H), 3.33−3.23 (m, 1H), 2.21−2.11 (m, 1H), 2.11−1.99 (m,
2H), 1.95−1.85 (m, 1H), 1.27−1.17 (m, 15H), 1.15−1.07 (m, 1H),
1
3
0.84 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 156.4,
3
2
2
154.1, 151.6, 139.5, 118.4, 58.4, 53.1, 38.4, 34.6, 29.64, 29.58, 29.5,
0:1) to give the target compound 6h as a white solid in 21.0 mg
29.4, 29.2, 26.4, 22.8, 14.2; ESI-HRMS (m/z) calcd C H ClN O
(M + H ), 368.2212; found, 368.2214.
1
8
31
5
1
+
3
6.1.78. 3-(6-Amino-2-chloro-9H-purin-9-yl)tetradecan-1-ol
(11h). 3-(2,6-Dichloro-9H- purin-9-yl)tetradecan-1-ol (40.1 mg, 0.1
mmol) (for 3-(2,6-dichloro-9H-purin-9-yl)tridecan-1-ol: prepared
according to the procedure for the synthesis of 9c, using 2,6-
dichloro-9H-purine to react with (E)-tetradec-2-enal) and ammonia
(2.0 mL, 7.0 M in MeOH) were added to a reaction tube with a
magnetic stirring bar. The reaction was stirred at 100 °C for 6 h and
detected by TLC. Then, the residue was concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
1
3
3
+
1
4
23
5
6
.1.74. 3-(6-Amino-2-chloro-9H-purin-9-yl)decan-1-ol (7h).
(CH
solid in 27.8 mg (73.0% yield). White solid. mp: 127−128 °C. H
NMR (400 MHz, CDCl
Cl /MeOH = 30:1) to give the target compound 11h as a white
2
2
1
) δ 7.78 (s, 1H), 6.80 (s, 2H), 4.71−4.61 (m,
3
1H), 3.61 (s, 2H), 3.34−3.22 (m, 1H), 2.22−2.10 (m, 1H), 2.10−
2
2
1.98 (m, 2H), 1.96−1.84 (m, 1H), 1.27−1.16 (m, 17H), 1.16−1.07
1
13
(m, 1H), 0.85 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl
) δ
3
156.6, 154. 0, 151.4, 139.6, 118.3, 58.3, 53.3, 38.2, 34.6, 32.0, 29.7,
3
29.6, 29.5, 29.4, 29.2, 26.4, 22.8, 14.2; ESI-HRMS (m/z) calcd
+
C
H
19
33ClN
O (M + H ), 382.2368; found, 382.2365.
5
1
3
6.1.79. 3-(8-Bromo-6-chloro-9H-purin-9-yl)octan-1-ol (5i). 8-
3
1
51.4, 139.5, 118.3, 58.3, 53.2, 38.2, 34.6, 31.8, 29.2, 29.1, 26.3, 22.7,
Bromo-6-chloro-9H-purine (46.6 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-oct-2-enal (44.8 μL, 0.3 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
+
14.2; ESI-HRMS (m/z) calcd C H ClN O (M + H ), 326.1742;
1
5
25
5
found, 326.1746.
6
.1.75. 3-(6-Amino-2-chloro-9H-purin-9-yl)undecan-1-ol (8h).
Compound 8c (35.9 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M
in MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
2
3
0:1) to give the target compound 8h as a white solid in 25.2 mg
1
(
72.2% yield). White solid. mp: 138−140 °C. H NMR (600 MHz,
CDCl ) δ 7.78 (s, 1H), 6.40 (s, 2H), 4.73−4.60 (m, 1H), 3.67−3.56
3
2
4
(
2
1
m, 1H), 3.37−3.01 (m, 2H), 2.25−2.11 (m, 1H), 2.11−1.98 (m,
column chromatography on silica gel (PE/EA = 3:1) to give the target
H), 1.98−1.86 (m, 1H), 1.27−1.24 (m, 5H), 1.23−1.19 (m, 6H),
.17−1.10 (m, 1H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (150 MHz,
compound 5i as a white solid in 27.8 mg (77.1% yield). White solid.
1
3
1
mp: 74−75 °C. H NMR (400 MHz, CDCl ) δ 8.65 (s, 1H), 4.88 (s,
3
CDCl ) δ 156.4, 154.1, 151.5, 139.5, 118.3, 58.4, 53.1, 38.3, 34.6,
1H), 3.69−3.61 (m, 1H), 3.39−3.29 (m, 1H), 2.66 (s, 1H), 2.50−
2.37 (m, 1H), 2.22−2.11 (m, 1H), 1.95−1.85 (m, 1H), 1.84−1.74
(m, 1H), 1.28−1.17 (m, 5H), 1.04−0.93 (m, 1H), 0.80 (t, J = 7.2 Hz,
3
3
1.9, 29.4, 29.23, 29.21, 26.4, 22.7, 14.2; ESI-HRMS (m/z) calcd
+
C H ClN O (M + H ), 340.1899; found, 340.1905.
16
27
5
1
3
6
.1.76. 3-(6-Amino-2-chloro-9H-purin-9-yl)dodecan-1-ol (9h).
3H); C NMR (150 MHz, CDCl ) δ 152.6, 151.4, 149.6, 136.3,
3
Compound 9c (37.3 mg, 0.1 mmol) and ammonia (2.0 mL, 7.0 M
in MeOH) were added to a reaction tube with a magnetic stirring bar.
The reaction was stirred at 100 °C for 6 h and detected by TLC.
Then, the residue was concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (CH Cl /MeOH =
132.2, 59.0, 58.2, 35.3, 33.0, 31.3, 26.0, 22.5, 14.0; ESI-HRMS (m/z)
+
calcd C H BrClN O (M + H ), 361.0425; found, 361.0430.
1
3
19
4
6.1.80. 3-(8-Bromo-6-chloro-9H-purin-9-yl)nonan-1-ol (6i). 8-
Bromo-6-chloro-9H-purine (46.6 mg, 0.2 mmol), L-proline (2.3 mg,
0.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
(E)-non-2-enal (49.7 μL, 0.3 mmol) was added to the reaction
2
2
3
0:1) to give the target compound 9h as a white solid in 26.2 mg
74.2% yield). White solid. mp: 46−147 °C. H NMR (400 MHz,
1
(
2
101
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109

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Article
mixture. The reaction was stirred at room temperature for 24 h and
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
detected by TLC. Then, the solution was cooled to 0 °C. After that,
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
column chromatography on silica gel (PE/EA = 3:1) to give the target
compound 9i as a white solid in 33.5 mg (80.6% yield). White solid.
2
4
1
column chromatography on silica gel (PE/EA = 3:1) to give the target
mp: 70−71 °C. H NMR (600 MHz, CDCl ) δ 8.64 (s, 1H), 4.85 (s,
3
compound 6i as a white solid in 29.7 mg (79.5% yield). White solid.
1H), 3.67−3.61 (m, 1H), 3.38−3.30 (m, 1H), 2.67 (s, 1H), 2.48−
2.39 (m, 1H), 2.20−2.11 (m, 1H), 1.95−1.80 (m, 2H), 1.26−1.20
(m, 4H), 1.20−1.14 (m, 9H), 1.02−0.93 (m, 1H), 0.83 (t, J = 7.2 Hz,
1
mp: 61−63 °C. H NMR (400 MHz, CDCl ) δ 8.64 (s, 1H), 4.87 (s,
3
1
2
H), 3.68−3.60 (m, 1H), 3.39−3.29 (m, 1H), 2.66 (s, 1H), 2.50−
.37 (m, 1H), 2.21−2.10 (m, 1H), 1.96−1.85 (m, 2H), 1.27−1.12
1
3
3H); C NMR (150 MHz, CDCl ) δ 152.5, 151.4, 149.6, 136.2,
3
1
3
(
m, 7H), 1.03−0.93 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H); C NMR
132.2, 58.9, 58.2, 35.3, 33.0, 31.9, 29.5, 29.4, 29.3, 29.1, 26.3, 22.7,
+
(
150 MHz, CDCl ) δ 152.5, 151.4, 149.6, 136.2, 132.2, 58.9, 58.2,
14.2; ESI-HRMS (m/z) calcd C H BrClN O (M + H ), 417.1051;
3
17 27
4
3
5.3, 33.0, 31.6, 28.8, 26.3, 22.6, 14.1; ESI-HRMS (m/z) calcd
found, 417.1058.
6.1.84. 3-(9H-Purin-9-yl)heptan-1-ol (4j). Compound 4b (26.9
+
C H BrClN O (M + H ), 375.0582; found, 375.0582.
14
21
4
6
.1.81. 3-(8-Bromo-6-chloro-9H-purin-9-yl)decan-1-ol (7i). 8-
mg, 0.1 mmol), Pd/C (10 wt %), and methanol (1.0 mL) were added
Bromo-6-chloro-9H-purine (46.6 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-dec-2-enal (54.7 μL, 0.3 mmol) was added to the reaction
to a reaction tube with a magnetic stirring bar under H (1 atm). The
2
0
reaction was stirred at room temperature until compound 4b was
completely consumed, which was detected by TLC. Then, the residue
was concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH Cl /MeOH = 30:1) to give the
(
mixture. The reaction was stirred at room temperature for 24 h and
2
2
detected by TLC. Then, the solution was cooled to 0 °C. After that,
target compound 4j as a colorless oil in 15.3 mg (65.8% yield).
1
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
Colorless oil. H NMR (400 MHz, CDCl ) δ 9.13 (s, 1H), 8.93 (s,
4
3
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
1H), 8.12 (s, 1H), 4.89−4.79 (m, 1H), 3.64−3.57 (m, 1H), 3.24−
3.08 (m, 2H), 2.26−2.07 (m, 3H), 2.05−1.92 (m, 1H), 1.33−1.20
1
3
(m, 3H), 1.15−1.02 (m, 1H), 0.82 (t, J = 7.2 Hz, 3H); C NMR
(150 MHz, CDCl ) δ 152.5, 151.8, 149.0, 144.4, 134.2, 58.4, 53.1,
3
38.0, 34.2, 28.6, 22.3, 13.9; ESI-HRMS (m/z), calcd C H N O (M
2
4
12 19
4
+
column chromatography on silica gel (PE/EA = 3:1) to give the target
+ H ), 235.1553; found, 235.1561.
compound 7i as a white solid in 32.2 mg (83.2% yield). White solid.
6.1.85. 3-(9H-Purin-9-yl)octan-1-ol (5j). Compound 5b (28.3 mg,
0.1 mmol), Pd/C (10 wt %), and methanol (1.0 mL) were added to a
1
mp: 60−62 °C. H NMR (600 MHz, CDCl ) δ 8.64 (s, 1H), 4.85 (s,
3
1
2
H), 3.67−3.61 (m, 1H), 3.37−3.30 (m, 1H), 2.67 (s, 1H), 2.49−
.38 (m, 1H), 2.20−2.12 (m, 1H), 1.96−1.84 (m, 2H), 1.31−1.25
reaction tube with a magnetic stirring bar under H (1 atm). The
2
reaction was stirred at room temperature until compound 5b was
completely consumed, which was detected by TLC. Then, the residue
was concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH Cl /MeOH = 30:1) to give the
(
m, 1H), 1.24−1.19 (m, 4H), 1.19−1.13 (m, 4H), 1.02−0.94 (m,
1
3
1
1
2
3
H), 0.81 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, CDCl ) δ 152.6,
3
51.4, 149.6, 136.1, 132.2, 59.0, 58.2, 35.3, 33.0, 31.7, 29.11, 29.05,
6.3, 22.6, 14.1; ESI-HRMS (m/z) calcd C H BrClN O (M + H ),
2
2
+
target compound 5j as a colorless oil in 15.6 mg (63.1% yield).
1
5
23
4
1
89.0738; found, 389.0740.
.1.82. 3-(8-Bromo-6-chloro-9H-purin-9-yl)undecan-1-ol (8i). 8-
Bromo-6-chloro-9H-purine (46.6 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-undec-2-enal (59.5 μL, 0.3 mmol) was added to the reaction
Colorless oil. H NMR (600 MHz, CDCl ) δ 9.17 (s, 1H), 8.96 (s,
3
6
1H), 8.13 (s, 1H), 4.94−4.75 (m, 1H), 3.67−3.54 (m, 1H), 3.27−
3.08 (m, 1H), 2.92 (s, 1H), 2.30−2.11 (m, 2H), 2.11−1.93 (m, 2H),
1
3
0
1.30−1.21 (m, 5H), 1.19−1.10 (m, 1H), 0.82 (t, J = 7.2 Hz, 3H); C
NMR (100 MHz, CDCl ) δ 152.5, 151.9, 149.1, 144.2, 134.3, 58.4,
3
(
52.9, 38.2, 34.4, 31.4, 26.1, 22.5, 14.0; ESI-HRMS (m/z) calcd
+
mixture. The reaction was stirred at room temperature for 24 h and
C H N O (M + H ), 249.1710; found, 249.1713.
1
3
21
4
detected by TLC. Then, the solution was cooled to 0 °C. After that,
6.1.86. 3-(9H-Purin-9-yl)nonan-1-ol (6j). Compound 6b (29.7 mg,
NaBH (22.7 mg, 0.6 mmol) was slowly added and the reaction was
0.1 mmol), Pd/C (10 wt %), and methanol (1.0 mL) were added to a
reaction tube with a magnetic stirring bar under an atmosphere of H2
(1 atm). The reaction was stirred at room temperature until
compound 6b was completely consumed, which was detected by
TLC. Then, the residue was concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (CH Cl /
4
stirred for 10 min. Then, the reaction was quenched with a saturated
ammonium chloride solution and concentrated in vacuo. Then, water
was added to the residue and the mixture was extracted with ethyl
acetate three times. The combined organic extracts were dried over
Na SO and concentrated in vacuo. The residue was purified by flash
2
4
2
2
column chromatography on silica gel (PE/EA = 3:1) to give the target
MeOH = 30:1) to give the target compound 6j as a colorless oil in
1
compound 8i as a white solid in 33.6 mg (83.5% yield). White solid.
17.4 mg (66.5% yield). Colorless oil. H NMR (400 MHz, CDCl ) δ
3
1
mp: 63−65 °C. H NMR (600 MHz, CDCl ) δ 8.66 (s, 1H), 4.86 (s,
9.18 (s, 1H), 8.97 (s, 1H), 8.13 (s, 1H), 4.92−4.81 (m, 1H), 3.66−
3.57 (m, 1H), 3.22−3.13 (m, 1H), 2.60 (s, 1H), 2.28−2.13 (m, 2H),
2.10−1.96 (m, 2H), 1.28−1.24 (m, 6H), 1.22−1.20 (m, 2H), 0.84 (t,
3
1
2
1
H), 3.68−3.62 (m, 1H), 3.38−3.31 (m, 1H), 2.68 (s, 1H), 2.50−
.40 (m, 1H), 2.20−2.13 (m, 1H), 1.95−1.87 (m, 1H), 1.63 (s, 1H),
.26−1.21 (m, 4H), 1.20−1.15 (m, 7H), 1.04−0.95 (m, 1H), 0.84 (t,
1
3
J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 152.5, 151.9, 149.1,
3
13
J = 7.2 Hz, 3H); C NMR (150 MHz, CDCl ) δ 152.6, 151.4, 149.7,
144.2, 134.3, 58.4, 52.9, 38.2, 34.5, 31.6, 28.9, 26.4, 22.6, 14.1; ESI-
3
+
1
36.2, 132.3, 59.0, 58.2, 35.3, 33.0, 31.9, 29.4, 29.21, 29.17, 26.3, 22.7,
HRMS (m/z) calcd C H N O (M + H ), 263.1866; found,
1
4
23
4
+
14.2; ESI-HRMS (m/z) calcd C H BrClN O (M + H ), 403.0895;
263.1865.
1
6
25
4
found, 403.0895.
6.1.87. 3-(6-Chloro-9H-purin-9-yl)-2-methyleneoctan-1-ol (5k).
6
.1.83. 3-(8-Bromo-6-chloro-9H-purin-9-yl)dodecan-1-ol (9i). 8-
Bromo-6-chloro-9H-purine (46.6 mg, 0.2 mmol), L-proline (2.3 mg,
.02 mmol), benzoic acid (2.4 mg, 0.02 mmol), and methanol (1.5
mL) were added to a reaction tube with a magnetic stirring bar. Then,
E)-dodec-2-enal (64.4 μL, 0.3 mmol) was added to the reaction
6-Chloro-9H-purine (0.93 g, 6 mmol), K CO (0.83 g, 6 mmol),
2
3
DABCO (0.022 g, 0.2 mmol), and methyl 2-methylene-3-
(propionyloxy) octanoate (0.48 g, 2 mmol) in CH Cl (15.0 mL)
0
2
2
were added to a reaction tube with a magnetic stirring bar under N2.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, to the mixture was added a saturated brine solution
and it was extracted with CH Cl three times. The combined organic
(
mixture. The reaction was stirred at room temperature for 24 h and
detected by TLC. Then, the solution was cooled to 0 °C. After that,
2
2
2
102
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Article
extracts were dried over Na SO and concentrated in vacuo. The
reaction was quenched with 10% NaOH solution. Then, the mixture
was extracted with CH Cl three times. The combined organic
2
4
residue was purified by flash column chromatography on silica gel
PE/EA = 2:1) to give the compound methyl 3-(6-chloro-9H-purin-9-
2
2
(
extracts were dried over Na SO and concentrated in vacuo. The
2 4
yl)-2-methyleneoctanoate as a colorless oil. Then, the solution of
methyl 3-(6-chloro-9H-purin-9-yl)-2-methyleneoctanoate (32.2 mg,
residue was purified by flash column chromatography on silica gel
(PE/EA = 1:1) to give the target compound 7k as a colorless oil in
1
0
.1 mmol) in CH Cl (1.0 mL) was cooled to −78 °C under N .
13.9 mg (43.2% yield). Colorless oil. H NMR (400 MHz, CDCl ) δ
2
2
2
3
DIBAL-H (0.3 mL, 0.3 mmol, 1.0 M in toluene) was added dropwise
and stirred at −78 °C for 20 min. The reaction was stirred at room
temperature until methyl 3-(6-chloro-9H-purin-9-yl)-2-methyleneoc-
tanoate was completely consumed, which was detected by TLC. The
reaction was quenched with 10% NaOH solution. Then, the mixture
was extracted with CH Cl three times. The combined organic
8.74 (s, 1H), 8.18 (s, 1H), 5.36 (s, 1H), 5.32 (t, J = 8.0 Hz, 1H), 5.25
(s, 1H), 4.14−4.01 (m, 2H), 2.38 (s, 1H), 2.27−2.18 (m, 2H), 1.33−
1
3
1.27 (m, 3H), 1.26−1.19 (m, 7H), 0.85 (t, J = 6.8 Hz, 3H); C NMR
(100 MHz, CDCl ) δ 152.02, 151.99, 151.3, 146.3, 144.2, 131.8,
3
114.9, 64.5, 56.4, 32.6, 31.8, 29.13, 29.08, 26.4, 22.7, 14.2; ESI-HRMS
+
2
2
(m/z) calcd C H ClN O (M + H ), 323.1633; found, 323.1642.
16 24
4
extracts were dried over Na SO and concentrated in vacuo. The
6.1.90. 3-(6-Chloro-9H-purin-9-yl)-2-methyleneundecan-1-ol
(8k). 6-Chloro-9H-purine (0.93 g, 6 mmol), K CO (0.83 g, 6
mmol), DABCO (0.022 g, 0.2 mmol), and methyl 2-methylene-3-
(propionyloxy)undecanoate (0.57 g, 2 mmol) in CH Cl (15.0 mL)
were added to a reaction tube with a magnetic stirring bar under N .
2
2
4
residue was purified by flash column chromatography on silica gel
PE/EA = 1:1) to give the target compound 5k as a colorless oil in
2
3
(
1
1
1.0 mg (37.7% yield). Colorless oil. H NMR (600 MHz, CDCl ) δ
2
2
3
8.75 (s, 1H), 8.18 (s, 1H), 5.36 (s, 1H), 5.36 (t, J = 7.2 Hz, 1H), 5.25
(
1
s, 1H), 4.13−4.01 (m, 2H), 2.28−2.18 (m, 3H), 1.27−1.23 (m, 5H),
1
3
.21−1.16 (m, 1H), 0.84 (t, J = 7.2 Hz, 3H); C NMR (150 MHz,
CDCl ) δ 152.0, 151.4, 148.8, 146.3, 144.1, 136.1, 115.0, 64.5, 56.4,
3
3
2.5, 31.3, 26.1, 22.5, 14.0; ESI-HRMS (m/z) calcd C H ClN O
1
4
20
4
+
(
M + H ), 295.1320; found, 295.1326.
.1.88. 3-(6-Chloro-9H-purin-9-yl)-2-methylenenonan-1-ol (6k).
-Chloro-9H-purine (0.93 g, 6 mmol), K CO (0.83 g, 6 mmol),
6
6
2
3
DABCO (0.022 g, 0.2 mmol), and methyl 2-methylene-3-
propionyloxy)nonanoate (0.51 g, 2 mmol) in CH Cl (15.0 mL)
(
2
2
were added to a reaction tube with a magnetic stirring bar under N2.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, to the mixture was added a saturated brine solution
and it was extracted with CH Cl three times. The combined organic
2
2
extracts were dried over Na SO and concentrated in vacuo. The
2
4
residue was purified by flash column chromatography on silica gel
PE/EA = 2:1) to give the compound methyl 3-(6-chloro-9H-purin-9-
(
The residue was purified by flash column chromatography on silica gel
(PE/EA = 1:1) to give the target compound 8k as a colorless oil in
1
12.4 mg (36.8% yield). Colorless oil. H NMR (600 MHz, CDCl ) δ
3
8.73 (s, 1H), 8.18 (s, 1H), 5.36 (s, 1H), 5.32 (t, J = 7.2 Hz, 1H), 5.24
(s, 1H), 4.12−4.02 (m, 2H), 2.47 (s, 1H), 2.26−2.18 (m, 2H), 1.32−
1.27 (m, 2H), 1.27−1.23 (m, 3H), 1.23−1.15 (m, 7H), 0.85 (t, J =
1
3
7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 152.03, 152.01, 151.4,
3
146.3, 144.1, 131.8, 114.9, 64.5, 56.4, 32.6, 31.9, 29.4, 29.23, 29.17,
+
26.4, 22.7, 14.2; ESI-HRMS (m/z) calcd C H ClN O (M + H ),
17 26
4
extracts were dried over Na SO and concentrated in vacuo. The
337.1790; found, 337.1795.
6.1.91. 3-(6-Chloro-9H-purin-9-yl)-2-methylenedodecan-1-ol
(9k). 6-Chloro-9H-purine (0.93 g, 6 mmol), K CO (0.83 g, 6
mmol), DABCO (0.022 g, 0.2 mmol), and methyl 2-methylene-3-
(propionyloxy)dodecanoate (0.60 g, 2 mmol) in CH Cl (15.0 mL)
were added to a reaction tube with a magnetic stirring bar under N .
2
2
4
residue was purified by flash column chromatography on silica gel
PE/EA = 1:1) to give the target compound 6k as a colorless oil in
(
1
2
3
1
2.7 mg (41.1% yield). Colorless oil. H NMR (400 MHz, CDCl ) δ
3
8
.72 (s, 1H), 8.18 (s, 1H), 5.36 (s, 1H), 5.20 (t, J = 8.0 Hz, 1H), 5.25
2
2
(
s, 1H), 4.13−4.01 (m, 2H), 2.58 (s, 1H), 2.27−2.18 (m, 2H), 1.34−
1
3
1
.26 (m, 3H), 1.26−1.17 (m, 5H), 0.84 (t, J = 6.8 Hz, 3H); C NMR
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, to the mixture was added a saturated brine solution
(100 MHz, CDCl ) δ 152.01, 151.97, 151.3, 146.2, 144.2, 131.7,
3
1
14.9, 64.4, 56.5, 32.6, 31.6, 28.8, 26.4, 22.6, 14.1; ESI-HRMS (m/z)
and it was extracted with CH
extracts were dried over Na
2
Cl
2
three times. The combined organic
and concentrated in vacuo. The
4
+
calcd C H ClN O (M + H ), 309.1477; found, 309.1473.
SO
2
15
22
4
6
.1.89. 3-(6-Chloro-9H-purin-9-yl)-2-methylenedecan-1-ol (7k).
residue was purified by flash column chromatography on silica gel
(PE/EA = 2:1) to give the compound methyl 3-(6-chloro-9H-purin-9-
yl)-2-methylenedodecanoate as a colorless oil. Then, the solution of
methyl 3-(6-chloro-9H-purin- 9-yl)-2-methylenedodecanoate (37.8
6
-Chloro-9H-purine (0.93 g, 6 mmol), K CO (0.83 g, 6 mmol),
DABCO (0.022 g, 0.2 mmol), and methyl 2-methylene-3-
propionyloxy)decanoate (0.54 g, 2 mmol) in CH Cl (15.0 mL)
2
3
(
2
2
were added to a reaction tube with a magnetic stirring bar under N2.
The reaction was stirred at room temperature for 24 h and detected
by TLC. Then, to the mixture was added a saturated brine solution
and it was extracted with CH Cl three times. The combined organic
mg, 0.1 mmol) in CH
2
Cl
2
(1.0 mL) was cooled to −78 °C under N .
2
DIBAL-H (0.3 mL, 0.3 mmol, 1.0 M in toluene) was added dropwise
and stirred at −78 °C for 20 min. The reaction was stirred at room
temperature until methyl 3-(6-chloro- 9H-purin-9-yl)-2-methylene-
dodecanoate was completely consumed, which was detected by TLC.
The reaction was quenched with 10% NaOH solution. Then, the
mixture was extracted with CH Cl three times. The combined
2
2
extracts were dried over Na SO and concentrated in vacuo. The
2
4
residue was purified by flash column chromatography on silica gel
PE/EA = 2:1) to give the compound methyl 3-(6-chloro-9H-purin-9-
(
2
2
yl)-2-methylenedecanoate as a colorless oil. Then, the solution of
organic extracts were dried over Na SO and concentrated in vacuo.
2 4
methyl 3-(6-chloro-9H-purin-9-yl)-2-methylenedecanoate (35.0 mg,
The residue was purified by flash column chromatography on silica gel
0
.1 mmol) in CH Cl (1.0 mL) was cooled to −78 °C under N .
(PE/EA = 1:1) to give the target compound 9k as a colorless oil in
2
2
2
1
DIBAL-H (0.3 mL, 0.3 mmol, 1.0 M in toluene) was added dropwise
and stirred at −78 °C for 20 min. The reaction was stirred at room
temperature until methyl 3-(6-chloro-9H-purin-9-yl)-2-methylenede-
canoate was completely consumed, which was detected by TLC. The
14.1 mg (40.1% yield). Colorless oil. H NMR (400 MHz, CDCl ) δ
3
8.72 (s, 1H), 8.18 (s, 1H), 5.36 (s, 1H), 5.32 (t, J = 8.0 Hz, 1H), 5.23
(s, 1H), 4.14−4.00 (m, 2H), 2.72 (s, 1H), 2.26−2.15 (m, 2H), 1.29−
1
3
1.09 (m, 14H), 0.85 (t, J = 6.8 Hz, 3H); C NMR (100 MHz,
2
103
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Article
replace racemic 9ba (124.8 mg, 0.3 mmol). [α]2 = −22.5 (c = 0.08,
0
3
D
CHCl ), 94% ee [CHIRALCEL IA, n-hexane/2-propanol = 98/2,
flow rate = 0.6 mL/min, temperature = 25 °C, λ = 250 nm, retention
time: 34.051 min (minor), 39.148 min (major)]. H NMR (400
3
1
MHz, CDCl ) δ 8.73 (s, 1H), 8.09 (s, 1H), 4.71−4.61 (m, 1H),
3
3.38−3.29 (m, 1H), 3.07−2.99 (m, 1H), 2.44−2.34 (m, 1H), 2.23−
2.09 (m, 2H), 1.99−1.88 (m, 1H), 1.27−1.15 (m, 13H), 1.09−0.98
1
3
(m, 1H), 0.85 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
152.0, 151.9, 151.4, 144.6, 132.2, 55.3, 48.1, 34.3, 33.7, 31.9, 29.5,
29.4, 29.3, 29.0, 26.2, 22.8, 14.2; ESI-HRMS (m/z) calcd C H ClN
1
7
27
7
+
(M + H ), 364.2011; found, 364.2014.
6.1.95. 6-Chloro-9-(1-(4-phenyl-1H-1,2,3-triazol-1-yl)dodecan-3-
yl)-9H-purine (9bc). Compound 9bb (36.3 mg, 0.1 mmol),
phenylacetylene (16.5 μL, 0.15 mmol), and Cu(OAc)2 (0.9 mg,
0.005 mmol) were added sequentially to acetonitrile (1.0 mL). The
mixture was stirred until Cu(OAc)2 was completely dissolved.
Subsequently, iPr NEt (1.7 μL, 0.01 mmol) was added and the
2
DIBAL-H (0.3 mL, 0.3 mmol, 1.0 M in toluene) was added dropwise
and stirred at −78 °C for 20 min. The reaction was stirred at room
temperature until methyl 2-((6-chloro-9H-purin-9-yl)(phenyl)-
methyl)acrylate was completely consumed, which was detected by
TLC. The reaction was quenched with 10% NaOH solution. Then,
the mixture was extracted with CH Cl three times. The combined
reaction mixture was stirred at 0 °C for 6 h. Then, water (2.0 mL) was
added to quench the reaction. The resulting mixture was extracted
with ethyl acetate three times. The organic phases were combined and
washed with saturated brine solution. The organic phase was dried
over anhydrous sodium sulfate and then concentrated in vacuo. The
residue was further purified by flash column chromatography on silica
gel (PE/EA = 3:1) to give the target racemic 9bc as a white solid in
32.8 mg (70.5% yield). White solid. mp: 98−100 °C. For (R)-9bc:
prepared according to the procedure for the synthesis of the above
racemic 9bc using (R)-9bb (36.3 mg, 0.1 mmol) to replace racemic
2
2
organic extracts were dried over Na SO and concentrated in vacuo.
2
4
The residue was purified by flash column chromatography on silica gel
PE/EA = 2:1) to give the target compound 10k as a white solid in
(
1
1
2.8 mg (42.8% yield). White solid. mp: 113−115 °C. H NMR (400
2
0
MHz, CDCl ) δ 8.72 (s, 1H), 7.95 (s, 1H), 7.48−7.38 (m, 3H),
7
9bb (36.3 mg, 0.1 mmol). [α] = −48.4 (c = 0.22, CHCl ), 94% ee
3
D
3
.38−7.28 (m, 2H), 6.62 (s, 1H), 5.51 (s, 1H), 4.70 (s, 1H), 4.21 (s,
H), 2.62 (s, 1H); C NMR (100 MHz, CDCl ) δ 152.2, 151.6,
[CHIRALCEL IA, n-hexane/2-propanol = 70/30, flow rate = 0.8 mL/
1
3
2
min, temperature = 25 °C, λ = 250 nm, retention time: 21.317 min
3
1
51.4, 145.9, 144.8, 134.8, 131.9, 129.6, 129.57, 128.6, 115.8, 64.4,
(major), 25.346 min (minor)]. H NMR (400 MHz, CDCl ) δ 8.73
3
+
(s, 1H), 8.16 (s, 1H), 7.80−7.74 (m, 2H), 7.67 (s, 1H), 7.42 (t, J =
7.2 Hz, 2H), 7.37−7.31 (m, 1H), 4.61−4.50 (m, 1H), 4.37−4.28 (m,
1H), 4.20−4.09 (m, 1H), 2.99−2.86 (m, 1H), 2.66−2.55 (m, 1H),
2.26−2.13 (m, 1H), 1.96−1.85 (m, 1H), 1.25−1.12 (m, 13H), 1.01−
1
5
14
4
found, 301.0855.
6
.1.93. 3-(6-Chloro-9H-purin-9-yl)dodecyl methanesulfonate
(
9ba). Compound 9b (169.1 mg, 0.5 mmol) and Et N (173.0 μL,
3
1
3
1
.25 mmol) were added to CH Cl (10.0 mL) at 0 °C.
0.90 (m, 1H), 0.84 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl )
2
2
3
Methanesulfonyl chloride (58.0 μL, 0.75 mmol) was slowly added
δ 151.9, 151.8, 151.6, 148.1, 145.2, 132.3, 130.3, 129.1, 128.5, 125.8,
to the solution. The mixture was stirred for 4 h at room temperature.
120.0, 55.6, 47.0, 34.7, 34.3, 31.9, 29.4, 29.31, 29.25, 29.0, 26.2, 22.7,
+
Then, the reaction was quenched with saturated NaHCO solution
14.2; ESI-HRMS (m/z) calcd C H ClN (M + H ), 466.2480;
3
25 33
7
and extracted with CH Cl . The organic phase was dried over
found, 466.2484.
2
2
anhydrous sodium sulfate and then concentrated in vacuo. The
residue was further purified by flash column chromatography on silica
gel (PE/EA = 2:1) to give the target compound 9ba as a white solid
in 157.6 mg (75.6% yield). White solid. mp: 73−74 °C. For (R)-9ba:
prepared according to the procedure for the synthesis of the above
racemic 9ba using (R)-9b (169.1 mg, 0.5 mmol) to replace racemic
6.1.96. 6-Chloro-9-(1-chlorododecan-3-yl)-9H-purine (9bd).
Compound 9b (169.1 mg, 0.5 mmol) and pyridine (44.5 μL, 0.55
mmol) were added to CH Cl (0.5 mL) at −20 °C. The reaction
2
2
mixture was stirred at −20 °C for 5 min. A solution of SO Cl (39.9
2
2
μL, 0.55 mmol) in CH Cl (0.5 mL) was slowly added dropwise. The
2
2
reaction mixture was stirred at −20 °C for 15 min and then at 0 °C
2
0
9
b (169.1 mg, 0.5 mmol). [α]D = −2.7 (c = 0.30, CHCl ), 94% ee
for another 15 min. After that, the CH Cl was evaporated and the
3
2
2
[
CHIRALCEL IA, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/
residue was suspended in diethyl ether. After filtration to remove the
precipitate, the organic phase was evaporated in vacuo. The residue
was further purified by flash column chromatography on silica gel
(PE/EA = 2:1) to give the target racemic 9bd as a colorless oil in
133.5 mg (75.0% yield). Colorless oil. For (R)-9bd: prepared
according to the procedure for the synthesis of the above racemic 9bd
using (R)-9b (169.1 mg, 0.5 mmol) to replace racemic 9b (169.1 mg,
min, temperature = 25 °C, λ = 250 nm, retention time: 47.048 min
1
(
minor), 51.301 min (major)]. H NMR (400 MHz, CDCl ) δ 8.71
3
(
s, 1H), 8.12 (s, 1H), 4.75−4.65 (m, 1H), 4.25−4.18 (m, 1H), 3.98−
3
2
0
.90 (m, 1H), 2.91 (s, 3H), 2.71−2.60 (m, 1H), 2.47−2.35 (m, 1H),
.24−2.14 (m, 1H), 2.00−1.88 (m, 1H), 1.26−1.13 (m, 13H), 1.07−
1
3
.95 (m, 1H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl )
3
2
0
δ 151.9, 151.8, 151.5, 145.0, 132.3, 65.8, 54.8, 37.6, 34.2, 33.9, 31.9,
9.5, 29.4, 29.3, 29.0, 26.2, 22.7, 14.2; ESI-HRMS (m/z) calcd
0.5 mmol). [α] = −21.8 (c = 0.15, CHCl ), 93% ee [CHIRALCEL
D
3
2
IA, n-hexane/2-propanol = 95/5, flow rate = 0.8 mL/min,
+
C H ClN O S (M + H ), 417.1722; found, 417.1719.
temperature = 25 °C, λ = 250 nm, retention time: 9.979 min
18
30
4
3
1
6
.1.94. 9-(1-Azidododecan-3-yl)-6-chloro-9H-purine (9bb). NaN3
(minor), 11.190 min (major)]. H NMR (400 MHz, CDCl ) δ 8.70
3
(
97.5 mg, 1.5 mmol) was added to a solution of racemic 9ba (124.8
(s, 1H), 8.09 (s, 1H), 4.81−4.70 (m, 1H), 3.55−3.46 (m, 1H), 3.18−
3.08 (m, 1H), 2.75−2.64 (m, 1H), 2.39−2.28 (m, 1H), 2.26−2.14
(m, 1H), 1.98−1.86 (m, 1H), 1.26−1.11 (m, 13H), 1.06−0.94 (m,
mg, 0.3 mmol) in DMF (5.0 mL) at 0 °C. Then, the reaction was
stirred at 25 °C for 6 h and detected by TLC. After that, water (10.0
mL) was added to quench the reaction. The resulting mixture was
extracted with ethyl acetate three times. The organic phases were
combined and washed with saturated brine solution. The organic
phase was dried over anhydrous sodium sulfate and then concentrated
in vacuo. The residue was further purified by flash column
chromatography on silica gel (PE/EA = 3:1) to give the target
racemic 9bb as a colorless oil in 81.1 mg (74.5% yield). Colorless oil.
For (R)-9bb: prepared according to the procedure for the synthesis of
the above racemic 9bb using (R)-9ba (124.8 mg, 0.3 mmol) to
1
3
1H), 0.82 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 151.8,
3
151.7, 151.4, 145.1, 132.3, 55.6, 41.0, 36.4, 33.8, 31.9, 29.4, 29.3, 29.2,
29.0, 26.2, 22.7, 14.2; ESI-HRMS (m/z) calcd C H Cl N (M +
1
7
27
2
4
+
H ), 357.1607; found, 357.1608.
6.1.97. Diethyl (((3-(6-Chloro-9H-purin-9-yl)dodecyl)oxy)-
methyl)phosphonate (9be). A solution of 9b (33.8 mg, 0.1 mmol)
in dry THF (0.5 mL) was slowly added to a stirred suspension of
NaH (12.0 mg, 0.5 mmol) in dry THF (0.5 mL) at −15 °C under N .
2
Then, the reaction mixture was stirred at −15 °C for 5 min. After that,
2
104
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Article
a solution of ((diethoxyphosphoryl)oxy) methyl trifluoromethanesul-
fonate (90.0 mg, 0.3 mmol) in THF (1.0 mL) was slowly added to the
above reaction mixture and the reaction mixture was stirred at 0 °C
for 7 h. Then, water (10.0 mL) was added to quench the reaction.
The resulting mixture was extracted with CH Cl three times. The
portion. The reaction was stirred at room temperature until
compound 17 was completely consumed, which was detected by
TLC. Then, the reaction mixture was quenched by adding solid
NaHSO to give a thick brown sludge. After filtering through a pad of
3
Celite using acetone/water as the eluent, a pale-yellow solution was
obtained, which was concentrated in vacuo to afford a white solid.
The residue was purified by flash column chromatography on silica gel
(CH Cl /MeOH = 20:1) to give the target compound 18 as a white
2
2
organic phases were combined and washed with a saturated brine
solution. The organic phase was dried over anhydrous sodium sulfate
and then concentrated in vacuo. The residue was further purified by
flash column chromatography on silica gel (CH Cl /MeOH = 90:1)
2
2
1
solid in 561.4 mg (83.5% yield). White solid. mp: 167−169 °C. H
2
2
to give the target racemic 9be as a colorless oil in 31.1 mg (63.8%
yield). Colorless oil. For (R)-9be: prepared according to the
procedure for the synthesis of the above racemic 9be using (R)-9b
NMR (400 MHz, CDCl ) δ 8.72 (s, 1H), 8.19 (s, 1H), 4.93−4.83 (m,
3
1H), 3.64 (s, 1H), 3.34 (dd, J = 17.2, 9.6 Hz, 1H), 2.97 (dd, J = 17.2,
4.0 Hz, 1H), 2.38−2.26 (m, 1H), 2.05−1.94 (m, 1H), 1.28−1.24 (m,
(
33.8 mg, 0.1 mmol) to replace racemic 9b (33.8 mg, 0.1 mmol).
4H), 1.23−1.17 (m, 9H), 1.11−1.00 (m, 1H), 0.86 (t, J = 7.2 Hz,
20
13
[
2
α] = −1.7 (c = 0.23, CHCl ), 92% ee [CHIRALCEL ID, n-hexane/
3H); C NMR (100 MHz, CDCl ) δ 173.2, 151.9, 151.5, 151.2,
D
3
3
-propanol = 80/20, flow rate = 0.8 mL/min, temperature = 25 °C, λ
250 nm, retention time: 19.747 min (minor), 21.282 min (major)].
145.9, 131.9, 55.1, 38.1, 33.3, 31.9, 29.5, 29.4, 29.3, 29.0, 26.3, 22.8,
+
=
14.2; ESI-HRMS (m/z) calcd C H ClN O (M + H ), 353.1739;
1
7
26
4
2
1
H NMR (600 MHz, CDCl ) δ 8.70 (s, 1H), 8.14 (s, 1H), 4.76−4.67
found, 353.1731.
3
(
m, 1H), 4.20−4.06 (m, 4H), 3.69−3.58 (m, 2H), 3.58−3.52 (m,
6.1.101. tert-Butyl (5-(3-(6-Chloro-9H-purin-9-yl)-
dodecanamido)pentyl)carbamate (20). To a stirred solution of
compound 18 (70.4 mg, 0.2 mmol) in CH Cl (2.0 mL) at 0 °C was
1
1
1
H), 3.24−3.12 (m, 1H), 2.44−2.34 (m, 1H), 2.28−2.16 (m, 2H),
.99−1.87 (m, 1H), 1.37−1.29 (m, 6H), 1.26−1.23 (m, 3H), 1.23−
2
2
1
3
added dropwise triethylamine (41.6 μL, 0.3 mmol). After 20 min,
isobutyl chloroformate (38.9 μL, 0.3 mmol) was added dropwise to
the above reaction mixture at 0 °C and the reaction was stirred for
another 30 min. Then, 5-(tert-butoxycarbonylamino)-pentyl amine
.16 (m, 10H), 1.06-0.97 (m, 1H), 0.85 (t, J = 7.2 Hz, 3H); C NMR
(
6
2
150 MHz, CDCl ) δ 151.9, 151.7, 151.2, 145.6, 132.3, 69.6, 69.6,
5.9, 64.8, 62.54, 62.50, 55.3, 34.2, 34.1, 31.9, 29.5, 29.4, 29.3, 29.1,
3
6.4, 22.8, 16.7, 16.6, 14.2; ESI-HRMS (m/z) calcd C H ClN O P-
2
2
39
4
4
+
(60.7 mg, 0.3 mmol) was added dropwise at 0 °C and the reaction
(
M + H ), 489.2392; found, 489.2396.
mixture was stirred for 20 min. The reaction was reacted at room
temperature until compound 18 was completely consumed, which
was detected by TLC. After that, water (2.0 mL) was added to
6
.1.98. Diethyl (((3-(6-Chloro-9H-purin-9-yl)dodecyl)oxy)-
methyl)phosphonic acid (9bf). To a solution of 9be (48.8 mg, 0.1
mmol) in anhydrous acetonitrile was added bromotrimethylsilane
(
132.0 μL, 1 mmol) at 0 °C. The reaction was stirred at room
quench the reaction. The reaction mixture was extracted with CH
three times. The combined organic extracts were dried over Na
2
Cl
SO
2
temperature overnight and detected by TLC. Then, the reaction
mixture was concentrated in vacuo. After that, water and CH Cl were
2
4
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
2
2
added. Then, the pH value of the mixture was adjusted to 7−8 by
adding 10% sodium hydroxide solution. Finally, the water phase was
concentrated in vacuo. The residue was purified by reversed phase
compound 20 as a colorless oil in 81.6 mg (76.1% yield). Colorless
1
oil. H NMR (600 MHz, CDCl
) δ 8.68 (s, 1H), 8.13 (s, 1H), 5.76 (s,
3
column chromatography (H O/MeOH = 3:1) to give the target
1H), 4.99−4.91 (m, 1H), 4.61 (s, 1H), 3.15−2.98 (m, 5H), 2.82 (dd,
2
compound 9bf as a white solid in 19.4 mg (44.9% yield). White solid.
J = 15.0, 4.8 Hz, 1H), 2.32−2.23 (m, 1H), 1.97−1.89 (m, 1H), 1.42
mp: >320 °C (Note: the melting point of 9bf exceeds the measuring
(s, 9H), 1.39−1.32 (m, 2H), 1.28−1.21 (m, 6H), 1.21−1.15 (m, 9H),
1
13
range of the thermometer). H NMR (400 MHz, D O) δ 8.85 (s,
1.10−1.03 (m, 2H), 1.03−0.96 (m, 1H), 0.84 (t, J = 7.2 Hz, 3H);
C
2
1
H), 8.70 (s, 1H), 3.64−3.52 (m, 1H), 3.51−3.32 (m, 3H), 2.60−
.36 (m, 2H), 2.26−2.11 (m, 1H), 2.10−1.95 (m, 1H), 1.25−1.07
NMR (151 MHz, CDCl ) δ 169.0, 156.3, 151.6, 151.5, 151.2, 146.4,
3
2
132.4, 79.3, 56.1, 40.5, 40.1, 39.3, 33.3, 31.9, 29.7, 29.5, 29.4, 29.3,
(
m, 2H), 1.06−0.96 (m, 1H), 0.93−0.84 (m, 5H), 0.83−0.70 (m,
29.0, 28.8, 28.6, 26.4, 23.7, 22.7, 14.2; ESI-HRMS (m/z) calcd
1
3
+
7
1
3
H), 0.51 (t, J = 7.2 Hz, 3H); C NMR (101 MHz, D O) δ 151.63,
C
H
27
46ClN
6
O
3
(M + H ), 537.3314; found, 537.3314.
2
49.13, 145.76, 141.24, 123.45, 69.43, 68.94, 68.84, 67.93, 53.98,
6.1.102. Biotin-Annexed Probe 21. A solution of 20 (80.4 mg, 0.15
3
1
4.53, 33.95, 31.09, 28.09, 28.01, 27.49, 24.81, 21.95, 13.40; P NMR
mmol) and CF COOH (2 mL) in CH Cl (2 mL) was stirred at
3
2
2
(
(
162 MHz, D O) δ 13.62; ESI-HRMS (m/z) calcd C H ClN O P
room temperature for 10 min. Then, the reaction mixture was
concentrated in vacuo to afford the corresponding deprotected amine.
To a solution of the above deprotected amine in anhydrous DMF (7
mL), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexa-
2
18 31
4
4
+
M + H ), 433.1766; found, 433.1773.
.1.99. 3-(6-Chloro-9H-purin-9-yl)dodecanal (17). 6-Chloro-9H-
6
purine (30.9 mg, 0.2 mmol), L-proline (2.3 mg, 0.02 mmol), benzoic
acid (2.4 mg, 0.02 mmol), and methanol (1.5 mL) were added to a
reaction tube with a magnetic stirring bar. Then, (E)-dodec-2-enal
fluorophosphate (HBTU, 56.9 mg, 0.15 mmol), iPr NEt (24.8 μL, 0.3
2
mmol), and a solution of D-(+)-biotin (24.4 mg, 0.1 mmol) in
anhydrous DMF (3 mL) were added subsequently. The reaction
(
64.4 μL, 0.3 mmol) was added to the reaction mixture. The reaction
was stirred at room temperature for 24 h and detected by TLC. Then,
the reaction was quenched with a saturated brine solution and
concentrated in vacuo. Then, water was added to the residue and the
mixture was extracted with ethyl acetate three times. The combined
organic extracts were dried over Na SO and concentrated in vacuo.
mixture was stirred at room temperature for 24 h under N and
2
detected by TLC. After that, water was added to the solution and the
reaction mixture was extracted with ethyl acetate three times. The
combined organic extracts were dried over Na
in vacuo. The residue was purified by flash column chromatography
on silica gel (CH Cl /MeOH = 12:1) to give the target compound 21
as a white solid in 23.8 mg (36.0% yield). White solid. mp: 54−56 °C.
SO and concentrated
2
4
2
4
The residue was purified by flash column chromatography on silica gel
PE/EA = 3:1) to give the target compound 17 as a white solid in
2
2
(
1
1
5
3.8 mg (80.1% yield). White solid. mp: 83−85 °C. H NMR (600
H NMR (400 MHz, DMSO-d ) δ 8.75 (s, 1H), 8.72 (s, 1H), 7.86 (t,
6
MHz, CDCl ) δ 9.70 (s, 1H), 8.70 (s, 1H), 8.15 (s, 1H), 5.03−4.96
J = 5.6 Hz, 1H), 7.66 (t, J = 5.6 Hz, 1H), 6.38 (s, 1H), 6.33 (s, 1H),
5.02−4.92 (m, 1H), 4.33−4.27 (m, 1H), 4.15−4.10 (m, 1H), 3.12−
3.06 (m, 1H), 2.98−2.77 (m, 8H), 2.57 (d, J = 12.4 Hz, 1H), 2.18−
2.07 (m, 1H), 2.03 (t, J = 7.2 Hz, 2H), 1.92−1.81 (m, 1H), 1.66−
1.56 (m, 1H), 1.54−1.40 (m, 4H), 1.26−1.23 (m, 4H), 1.18−1.11
3
(m, 1H), 3.55 (dd, J = 18.6, 8.4 Hz, 1H), 3.12 (dd, J = 18.6, 4.8 Hz,
1
1
H), 2.29−2.21 (m, 1H), 1.96−1.88 (m, 1H), 1.28−1.21 (m, 4H),
13
.21−1.15 (m, 9H), 1.05−0.98 (m, 1H), 0.84 (t, J = 7.2 Hz, 3H);
C
NMR (150 MHz, CDCl ) δ 198.3, 151.6, 151.5, 151.3, 145.6, 132.3,
3
5
2.4, 47.5, 33.5, 31.9, 29.5, 29.3, 29.3, 28.9, 26.2, 22.7, 14.2; ESI-
(m, 13H), 1.06−0.99 (m, 2H), 0.98−0.90 (m, 1H), 0.82 (t, J = 6.8
+
13
HRMS (m/z) calcd C H ClN O (M + H ), 337.1790; found,
Hz, 3H); C NMR (100 MHz, DMSO-d ) δ 171.7, 168.5, 162.6,
1
7
26
4
6
3
37.1785.
151.8, 151.1, 149.0, 147.0, 131.0, 61.0, 59.2, 55.4, 54.1, 38.2, 35.2,
33.1, 31.2, 28.7, 28.6, 28.5, 28.5, 28.1, 28.0, 25.3, 23.5, 22.0, 13.9; ESI-
6
.1.100. 3-(6-Chloro-9H-purin-9-yl)dodecanoic Acid (18). To a
+
solution of compound 17 (674.4 mg, 2.0 mmol) in acetone (5 mL)/
HRMS (m/z) calcd C H ClN O S (M + H ), 663.3566; found,
3
2
52
8
3
water (2 mL) was added KMnO (632.1 mg, 4.0 mmol) in one
663.3571.
4
2
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Article
6
.1.103. Diazirine Photoaffinity Probe 23. To a stirred solution of
transferred onto a poly(vinylidene fluoride) (PVDF) membrane. After
blocking with 5% BSA in TBST, the membrane was interacted with
GAPDH, Bax, P53, Bcl-xl, and cleaved-PARP primary antibody
overnight at 1:2000 dilutions. Then, the membrane was interacted
with appropriate secondary antibodies conjugated with horseradish
peroxidase (HRP) at room temperature for 2 h. Protein bands are
visualized by chemiluminescence reagents.
compound 18 (70.4 mg, 0.2 mmol) in CH Cl (2.0 mL) at 0 °C was
2
2
added dropwise triethylamine (41.6 μL, 0.3 mmol). After 20 min,
isobutyl chloroformate (38.9 μL, 0.3 mmol) was added dropwise to
the above reaction mixture at 0 °C and the reaction was stirred for
another 30 min. Then, 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-
amine (38.4 μL, 0.3 mmol) was added dropwise at 0 °C and the
reaction mixture was stirred for 20 min. The reaction was reacted at
room temperature until compound 18 was completely consumed,
which was detected by TLC. After that, water (2.0 mL) was added to
quench the reaction. The reaction mixture was extracted with CH Cl
5
6.6. MMP Determination. Around 5 × 10 SW480 cells per well
were seeded into a six-well plate for 24 h at 37 °C. Then, cells were
treated with diluent (DMSO) or specified concentrations of
compound 9b for 24 h. For flow cytometer detection, cells were
collected with a trypsin solution, washed twice with PBS, followed by
adding JC-1 binding solution, and then incubated in the incubator at
37 °C for 20 min in a dark place, centrifuged, and washed 3 times
with JC-1 staining buffer. After resuspending with JC-1 staining buffer,
the samples were analyzed with a flow cytometer. For fluorescence
detection, cells were washed twice with PBS, stained with the JC-1
binding solution at 37 °C for 20 min in a dark place, and washed
twice with JC-1 staining buffer. Images were taken under a
fluorescence microscope.
2
2
4
three times. The combined organic extracts were dried over Na SO
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (PE/EA = 1:1) to give the target
2
compound 23 as a colorless oil in 83.8 mg (88.9% yield). Colorless
1
oil. H NMR (600 MHz, CDCl ) δ 8.66 (s, 1H), 8.11 (s, 1H), 5.84−
3
5
2
1
1
1
.78 (m, 1H), 4.96−4.89 (m, 1H), 3.15 (dd, J = 15.6, 9.6 Hz, 1H),
.99−2.92 (m, 1H), 2.90−2.84 (m, 1H), 2.81 (dd, J = 15.0, 4.8 Hz,
H), 2.32−2.23 (m, 1H), 1.95 (t, J = 2.5 Hz, 1H), 1.95−1.90 (m,
H), 1.90−1.83 (m, 2H), 1.53−1.38 (m, 4H), 1.25−1.17 (m, 5H),
1
3
.17−1.12 (m, 8H), 1.02−0.94 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H);
NMR (151 MHz, CDCl ) δ 169.07, 151.58, 151.44, 151.09, 146.28,
32.41, 82.70, 69.52, 55.90, 40.33, 34.40, 33.19, 32.13, 31.90, 31.82,
9.40, 29.31, 29.21, 28.93, 26.64, 26.30, 22.65, 14.12, 13.14; ESI-
HRMS (m/z) calcd C H ClN O (M + H ), 472.2586; found,
72.2585.
6
dissolved in dimethyl sulfoxide (DMSO, Kermel) at a concentration
of 100 mM and stored at 4 °C. Dulbecco’s modified Eagle’s medium
DMEM), RPMI-1640 medium, McCoy’s 5A medium, phosphate
C
6.7. Colony Formation Assay. HCT-116 cells and SW480 cells
were seeded in six-well plates with a density of 1500 cells per well and
maintained in a regular culture medium. After 24 h, the cells were
treated with compound 9b at indicated concentrations for 48 h. The
culture medium was changed every 72 h. At the end of 14 days, the
wells were washed twice with PBS, fixed with methanol for 20 min,
and stained with 2 mL of crystal violet staining solution for 30 min at
room temperature. The wells were then washed with PBS three times
and allowed to dry. Photographs were then taken.
3
1
2
+
2
4
35
7
4
.2. Reagents and Antibodies. These compounds were
(
6.8. Metabolic Stability Assay. Microsomes were dissolved in
buffer (PBS), and fetal bovine serum (FBS) were purchased from
ThermoFisher Scientific. Poly(vinylidene fluoride) (PVDF) mem-
brane was purchased from Millipore. Bax, Bcl-xl, and cleaved-PARP
primary antibodies were purchased from Proteintech; antirabbit or
antimouse IgG horseradish peroxidase (HRP) -linked secondary
antibodies were purchased from Arigo (China); the cell apoptosis
detection kit was purchased from BD Biosciences; P53 and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary anti-
bodies, crystal violet staining solution, BCA protein assay kit, IP lysis
buffer, mitochondrial membrane potential assay kit, and cell-cycle
analysis kit were purchased from Beyotime (China); and Q-VD-OPh
was purchased from MedChemExpress.
0.1 M Tris buffer (pH 7.40) containing 5 mM MgCl , 0.1 μM
2
compound 9b, or 0.01% DMSO (negative control) and 0.005%
bovine serum albumin (BSA) at the concentration of 0.33 mg/mL
and incubated at 37 °C for 10 min. The reaction was started by the
addition of NADPH (final concentration 1 mM). Aliquots were
sampled at 0, 7, 17, 30, and 60 min, and methanol (cold in 4 °C) was
added to terminate the reaction. After centrifugation (4000 rpm, 5
min), samples were then analyzed by LC-MS/MS.
6.9. Acute Toxicity Assay. Balb/C mice were divided into six
groups randomly (n = 10, five male and five female) and injected
intraperitoneally with various dosages of compound 9b (75, 62.5, 50,
37.5, or 25 mg/kg) or a diluent (DMSO/castor oil/PBS = 1:1:8) per
4 h; after 4 times of injection, mice were fed for 14 days and the
number of dead mice were recorded every day. The LD50 value was
evaluated in GraphPad Prism 6.0.
6.3. MTT Cell Viability Assay. The antiproliferation activities of
synthesized compounds were assessed by an MTT assay. Cells were
cultured in DMEM, RPMI-1640, or McCoy’s 5A medium
supplemented with 10% FBS and 1% penicillin-streptomycin solution
6.10. Xenograft Tumor Growth Assay in Nude Mice. All
animal studies and procedures were approved by the Animal Ethics
Committee of School of Pharmacy, Fudan University. BALB/C nude
mice at 5 weeks of age were purchased from Shanghai Slac Laboratory
Animal Co. Ltd. (Shanghai, China) and housed under a specific
pathogen-free environment in an animal holding unit (AHU) of
Fudan University (Shanghai, China). Mice were injected with SW480
(
10 000 U/mL penicillin and 10 000 μg/mL streptomycin) at 37 °C
3
under 5% CO . Then, 5 × 10 cells per well were seeded into 96-well
2
plates for 24 h and then treated with compounds at various
concentrations or DMSO (diluent) for 48 h. Then, 20 μL of MTT
solution (5 mg/mL in PBS) was added to each well and incubated at
3
7 °C for 4 h. Next, the MTT solution was replaced with DMSO (150
μL). Absorbance of each well was tested by a microplate reader
Multiskan FC, Thermo) at a 570 nm wavelength. Assays were
6
cells (4 × 10 per mouse) subcutaneously. Seven days after injection,
(
mice were randomly divided into four groups (n = 8) and
administered intraperitoneally with 25 or 50 mg/kg compound 9b
(dissolved in PBS containing 5% ethanol and 5% cremophor EL, 0.2
mL per mouse), 5-FU at 50 mg/kg, or vehicle control (0.2 mL of the
diluent) once every 2 days for 12 days. At the end of the experiment,
the mice were weighed and sacrificed and tumors were collected and
fixed with 4% paraformaldehyde and embedded in paraffin. H & E
staining was used to further analyze the solid tumors of mice.
6.11. Pull-Down Assay. The biotin-annexed probe 21 or the
diazirine-based probe 23 was incubated with SW480 cell lysate at 4
°C overnight. The streptavidin conjugate beads (30 μL) were added
and incubated for 8 h at 4 °C. Then, the beads were washed 10 times
with Western and IP cell lysis buffer. After that, the Western and IP
cell lysis buffer (20 μL) and the loading buffer (5 μL) were added and
boiled for 10 min. For the diazirine-based probe 23, after 20 min of
irradiation at 360 nm, the probe 23 was incubated with the azide−
PEG3−biotin conjugate for 3 h at 4 °C. After that, it was incubated
performed in triplicates. Data were analyzed by GraphPad Prism 6.
Data are mean ± SEM values from three independent experiments.
5
6
.4. Annexin V-FITC/PI Apoptosis Assay. Around 5 × 10
SW480 cells per well were seeded into six-well plates, incubated
overnight, and treated with compound 9b at specified concentrations
for 48 h. Then, the cells were collected, washed twice with PBS, and
costained with Annexin V-FITC and PI in a dark place for 15 min at
room temperature according to the kit manual. Apoptotic cells were
detected with flow cytometry, and 10 000 cells were counted each
time.
6.5. Western Blotting Assay. SW480 cells were seeded into six-
well plates, incubated overnight, and treated with compound 9b at
specified concentrations for 48 h. The cells were collected, and the
cell samples were lysed by IP lysis buffer. Protein concentrations were
quantified by the BCA protein assay kit. Cell extracts were separated
by electrophoresis on sodium dodecyl sulfate polyacrylamide gels and
2
106
https://dx.doi.org/10.1021/acs.jmedchem.0c01717
J. Med. Chem. 2021, 64, 2077−2109