Syntheses and biological evaluation of novel hydroxamic acid derivatives containing purine moiety as histone deacetylase inhibitors

Article abstract of DOI:10.1248/cpb.c17-00997

The novel hydroxamates containing purine scaffold were designed, synthesized and screened for their biological activities as histone deacetylase (HDAC) inhibitors. Some of them exhibited excellent acti-HDACs activities and antiproliferative activities, th

Full text of DOI:10.1248/cpb.c17-00997

Vol. 66, No. 4
Chem. Pharm. Bull. 66, 439–451 (2018)
439
Regular Article
Syntheses and Biological Evaluation of Novel Hydroxamic Acid
Derivatives Containing Purine Moiety as Histone Deacetylase Inhibitors
Zhaoxing Xu,# Yongchao Yang,# Xi Mai,* Bin Liu, Yuanzhen Xiong, Lihuang Feng, Yijing Liao,
Yu Zhang, Huanlu Wang, Leiting Ouyang, and Shuhao Liu
School of Pharmaceutical Sciences, Nanchang University; Nanchang 330006, P. R. China.
Received December 11, 2017; accepted January 25, 2018
The novel hydroxamates containing purine scaffold were designed, synthesized and screened for their
biological activities as histone deacetylase (HDAC) inhibitors. Some of them exhibited excellent acti-HDACs
activities and antiproliferative activities, the most promising compound was 7mꢀ. Western blot analysis indi-
cated the compounds 7fꢀ, 7lꢀ, 7mꢀ, 7oꢀ could increase histone H3 acetylation levels in HCT116 and K562 cell
lines, and 7mꢀ increased the level of acetyl histone H3 in a dose-dependent manner, which is similar to the
behavior of suberoylanilide hydroxamic acid (SAHA). Molecular docking study revealed that the conforma-
tion of 7mꢀ in the active site of HDAC2 was similar to positive drug SAHA, which were oriented with the
hydroxamic acid towards the catalytic center and formed metal binding with zinc ion.
Key words hydroxamic acid; purine; histone deacetylase; antiproliferative; Western blot analysis
At present a large number of researches indicate that tu- with the amino acid residues on the rim of the enzymatic ac-
morigenesis is closely related to histone deacetylases (HDACs) tive site, joined by a linker domain with proper length.18,19)
because they trigger the abnormal transcription of crucial
Many purine compounds have been described as anti-
genes that control essential cell functions, namely prolifera- cancer derivatives, such as early 6-mercaptopurine have been
tion, cell cycle regulation and apoptosis.1,2) It also imparts its extensively used in clinical as an anti-tumor drug through
role in several genome functions such as DNA repair, chroma- inhibiting the synthesis of nucleic acid in tumor cells,20,21) and
tin assembly and recombination. Nearly 18 HDAC isoforms then Nelarabine, Fludarabine, Cladribine, Clofarabine were
have been identified in humans. They have been categorized discovered as anti-tumor drugs.22–25) Substituted at 2, 6, 8 and
into five classes based on their cellular location, size, number 9 positions have been the focus of structural modifications of
of catalytic pockets and homology to yeast prototypes.3,4) The the purine ring. Both mono-substituted and 2, 6, 8, 9 posi-
enzymes of classes I (HDAC 1, 2, 3 and 8), IIa (HDACs 4, tions multi-substituted derivatives show multiple mechanism
5, 7, 9), IIb (HDACs 6, 10) and IV (HDAC 11) are all Zn2+- of action.26–30) In consideration of excellent characteristics of
dependent metalloproteases, whereas the class III HDACs purine derevatives on anti-tumor, the combination of purine
(SIRT1-7) are oxidized form of nicotinamide adenine di- and hydroxamic acid was expected to exert their respective
nucleotide (NAD+) dependent.5–7) It has been suggested that superiority, the novel series of purine-containing hydroxamic
Zn2+-dependent HDACs, especially class I and class II iso- acids were designed and synthesized, wherein purine was
zyme, are closely related to tumorigenesis and development, selected as the cap domain based on the fact that most potent
and inhibition of HDACs can result in proliferation inhibition, HDACi possessed aromatic or heteroaromatic rings in their
apoptosis, cellular differentiation and migration inhibition of cap groups,12,31) expecting that purine scaffold could not only
tumor cells.8–10) Therefore, HDACs inhibitors (HDACi) against form special interation with the rim of the enzymatic active
Zn2+-dependent HDACs have been developed extensively.11,12) site but also exert synergistic effects on anti-tumor due to
Over the past years, the U.S Food and Drug Administration its multiple biological activities. Of target compounds, one is
has approved several HDACi, including suberoylanilide hy- both 6-substituted purine hydroxamic acids and 6-substituted
droxamic acid (SAHA, Vorinostat) and FK-228 (romidepsin) purine hydroxycarbamides containing hydroxyurea pharma-
for the treatment of refractory cutaneous T-cell lymphoma,13,14) cophore. Our group has made great efforts to develop novel
PXD101 (belinostat) for treatment of refractory bperipheral hydroxycarbamides to evaluate their anticancer activity,32–35)
T-cell lymphoma,15) and LBH589 (panobinostat) for the treat- and found that some hydroxycarbamides possessed potent
ment of multiplemyeloma.16) Because of strong zinc-chelating antitumor activity. To explore the possibility of synergistic
ability, hydroxamic acid remains among the most potent and effects, we introduced aliphatic diamines to 6-position of
popular zinc ion binding group (ZBG) reported for inhibition purine as linker and synthesized 6-substituted purine hydroxy-
of Class I HDACs.17) Among approved drugs, SAHA, PXD101, carbamides, namely, 6-substituted purine hydroxamic acids. In
LBH589 both possess a hydroxamic acid moiety. Despite the addition, based on the aliphatic carbonchain linker of SAHA,
variety of structural characteristics, most HDACs inhibitors, we introduced aliphatic carbonchain to 9-position of purine
including hydroxamates, can be considered to have a common and synthesized 9-substituted purine hydroxamic acids (Fig.
pharmacophore, which mainly contains three parts: a zinc ion 1). The HDACs inhibitory activities, anticancer activities and
binding group (ZBG) and a cap group which makes contacts effect of histone H3 acetylation of these novel compounds
were comprehensively investigated in this paper.
# These authors contributed equally to this work.
*To whom correspondence should be addressed. e-mail: maixi123@aliyun.com
© 2018 The Pharmaceutical Society of Japan

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Fig. 1. Design Strategy of Purine-Containing Hydroxamic Acid Derivatives
Reagents and conditions: (i) Diamine, TEA, n-butanol, reflux, 6h; (ii) TFA, aromaticamine, 120°C, 12h. (iii) 4-Nitrophenyl chloroformate, NaHCO3, H2O: aceto-
nitrile=2:3, 0°C, 0.5–1h. (iv) NH2OH · HCl, NaOH, MeOH, 55°C, 5h.
Chart 1
Reagents and conditions: (v) Amines, TEA, n-butanol, reflux, 6h. (vi) Ethyl 4-bromobutanoate (or Ethyl 8-bromooctanoate), K2CO3, DMF, 25°C, overnight. (vii)
NH2OH·HCl, CH3ONa, MeOH, 12h.
Chart 2

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Chem. Pharm. Bull.
441
Table 1. The HDACs Inhibitory Activities of Compounds
give 3a–3l,33,34) and then 3a–3l were converted to target com-
Results and Discussion
Chemistry The starting materials 6-chloro-9H-purine, pounds 4a–4l in the presence of NaOH in anhydrous methanol
6-chloro-9H-purin-2-amine and 2,6-dichloro-9H-purine are with hydroxylamine hydrochloride (Chart 1).
commercially available. The target compounds 4a–4l and
7a′–7p′ were synthesized following the procedures described chloro-9H-purine and different aliphatic amine and aromatic
in Chart 1 and 2. amine, their synthesis methods were same as that of 1a–1d. In
Intermediates 5 could be obtained by reaction of 2,6-di-
The intermediates 1a–1g, 1j were synthesized through a nu- the presence of K2CO3, compounds 5 were treated with ethyl
cleophilic substitution reaction of starting materials with dif- 4-bromobutanoate or ethyl 8-bromooctanoate in DMF at room
ferent diamine in n-butanol. Intermediates 2g–2l, which were temperature for overnight to give 6a′–6p′, and then converted
substituted by aromatic amine at position 2, were synthesized to target compounds 7a′–7p′ in the presence of CH3ONa in
using 1g and 1j, which should be catalyzed by trifluoroacetic anhydrous methanol with hydroxylamine hydrochloride (Chart
acid (TFA). In the presence of NaHCO3, compounds 1a–1f 2).
and 2g–2l were treated with 4-nitrophenyl chloroformate to
HDACs Inhibition Assays In vitro bioactivity evaluation

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of compounds 4a–4l and 7a′–7p′ were performed by HDACs HDACs activities. On the other hand, the length of the linker
activity assays using an HDAC Colorimetric Assay/Drug Dis- had also an effect on anti-HDACs activities. Using longer
covery kit (AK501, Enzo Biochem Inc.) (mainly HDAC 1&2). linkers (compounds 7j′–7p′) in contrast to shorter linkers ana-
Assays were performed according to its product manual, and logs (compounds 7a′ and 7d′–7i′) would increase the biologi-
SAHA was used as positive control. The test results were pre- cal activities, and so were compouns 4a–4d, their activities
sented in Table 1.
increased with the extension of linker.
As shown in Table 1, compounds 4d, 7c′, 7f′, 7h′, 7m′, 7o′
Docking Study Docking studies were performed to gain
and 7p′ showed comparable inhibitory activity with SAHA insight into the protein–inhibitor interactions within the
and PXD101. The majority of the compounds 7 showed in- enzyme binding sites. The crystal structure of HDAC2 in
teresting activity, among them, four compounds 7m′, 7p′, 7f′ complex with representative hydroxamate SAHA was used for
and 7o′ exhibited very potent inhibitory activities with the docking experiments of target compounds.36) The 3D crystal
IC50 values of 15.3, 16.4, 17.8 and 18.1nM. Compounds 4g–4l, structure of HDAC2 (PDB ID: 4LXZ) was collected from
which possesses aromatic groups at position 2 of purine, RCSB-Protein Data Bank (RCSB-PDB). The docking mode
showed poor activities, suggesting the substitution with bulky comparison of the most active compound 7m′ and reference
groups on purine C2-position was unfavorable for the in- drug SAHA in the active site of HDAC2 was demonstrated
hibitory activities. In general, compounds 7 bearing aliphatic in Fig. 2. The result suggested that compound 7m′ could in-
carbonchain linkers were shown to be more potent aganist teract with catalytic pocket and bind similarly to SAHA in
HDACs than compounds 4 bearing aliphatic diamines linkers, the active site of HDAC2. The hydroxamate part of 7m′ and
suggesting 9-substituted purine hydroxamic acids may be su- SAHA could chelate zinc ion in a similar trigonal bipyrami-
perior to the 6-substituted purine hydroxycarbamides in anti- dal fashion, with virtrally identical positioning of the Zn2+
interacting functional groups. Except for metal binding, the
generally observed interations of 7m′ with HDAC2 involved
hydrogen bonds and hydrophobic interations. As illustrated in
Fig. 3 for 7m′ docked into the enzymatic cavity of HDAC2
via Ligplot program,37) 7m′ could form hydrogen bond with
amino acid residue His145, His146, Tyr308, His183, Asp181,
Phe210 and hydrophobic interactions with Gly154, Phe155,
Leu276, Tyr209, Glu208, Gly212. Comparison with the SAHA
docking shows that both of them could form similar hydrogen
bonds with amino acid residues His145, His146, Tyr308, and
similar hydrophobic interactions with Phe155 and Gly154. The
Phe155 is a residue in the rim of the active pocket entrance,
SAHA and 7m′ interacted with Phe155 by phenyl ring and pu-
ring ring, respectively, indicating puring as cap domain could
increased binding of target compounds with the rim of active
pocket similar to the phenyl ring of SAHA.
Antitumor Evaluation With the aim to estimate the
potential antiproliferative activity of compounds 7, we have
Fig. 2. The Docking Mode Comparison of Compounds SAHA, 7m′
evaluated their cytotoxicity against two human cancer cell
Fig. 3. The 2D Interation Diagrams of 7m′ (A) and SAHA (B) with HDAC2 Active Site
The ligands and protein side chains are shown in ball-and-stick representation, the spoked arcs represent protein residues making hydrophobic contacts with the ligand.

Vol. 66, No. 4 (2018)
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443
lines, namely HCT116 (colon carcinoma cell line) and K562 exhibited better anti-proliferative activity than the compounds
(leukemia cancer cell line) by the improved 3-(4,5-dimeth- 7a′–i′ with short linker, these findings were consistently with
ylthiahiazol-2-y1)-2,5-diphenytetrazolium bromide (MTT) the results of the HDACs inhibition results.
assay32) and SAHA, PXD101 were used as positive control, re-
Upregulation Effect of Histone Acetylation Levels To
sults were presented in Table 2. Out of the tested compounds, determine whether the target compounds increase histone
most of compounds 7, with the only exception of 7i′, inhibited acetylation levels, compounds 7m′, 7f′, 7l′, 7o′ were tested for
HCT116 cells with IC50 values from 0.13 to 62.16µM, and 7b′, their effects on histone H3 acetylation levels in HCT116 and
7f′, 7j′, 7l′, 7m′ proved more potent than positive drug SAHA, K562 cells (Fig. 4), using SAHA as reference compounds.
PXD101. Notably, 7m′ was the most potent compound against The results showed that at 10µM each of the above compounds
HCT116 cells with an IC50 value of 0.13µM. Regarding their could increase the level of acetylated histone H3 in two cell
activity against K562 cells, twelve compounds (7e′–7p′) lines, which were similar to the behavior of SAHA. Among
showed IC50 values between 1.30 and 40.69µM, among them, four tested compounds, the effect of 7m′ on the acetylation
7f′, 7l’, 7m′ proved more potent than positive drug PXD101 degree of histone H3 was the best, especially the effect of
with IC50 values of 4.68, 5.27, 1.30µM, respectively. Most of 7m′ on HCT116 was higher than that of SAHA. The does-
the tested compounds, except for 7e′, 7h′ and 7p′, exhibited dependencies of 7m′ and SAHA on histone acetylation were
higher activities on HCT116 cells than K562 cells, which may evaluated (Fig. 5), the results showed that 7m′ could increase
be due to the higher HDACs expression level in HCT116 the amount of acetylated histone H3 in a dose-dependent man-
cells.38) In general, the compounds 7j′–p′ with long linker ner, which is similar to the behavior of SAHA.
Table 2. The Antiproliferative Activities of Compounds
IC50 (µM)
Compounds
HCT116
K562
7a′
7b′
7c′
7d′
7e′
7f′
7g′
7h′
7i′
7j′
7k′
7l′
7m′
7n′
7o′
7p′
SAHA
PXD101
7.59 0.62
1.07 0.05
5.52 1.15
24.05 5.50
62.16 7.70
1.25 0.16
3.11 0.41
20.21 2.31
>120
0.64 0.03
3.04 0.08
1.39 0.04
0.13 0.01
10.45 3.01
14.45 1.01
12.54 6.92
1.55 0.28
3.17 0.60
111.27 1.14
>120
>120
98.34 0.33
37.53 2.21
4.68 0.12
21.59 0.12
20.29 0.91
18.07 0.71
26.79 1.01
40.69 1.26
5.27 0.51
1.30 0.012
12.82 0.43
11.58 0.19
25.38 0.88
0.56 0.39
7.45 0.52
Fig. 4. Effect of Histone H3 Acetylation of 7m′, 7f′, 7l′, 7o′ at 10µM
in Cultured HCT116 Cancer and K562 Cancer Cells by Western Blotting
Fig. 5. Effect of Histone H3 Acetylation of 7m′, SAHA at Different Concentrations in Cultured HCT116 Cancer and K562 Cancer Cells by Western
Blotting

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J=6.0Hz, 2H, CH2), 8.07 (s, 1H, CH), 8.17 (s, 1H, CH) ppm.
N 1-(9H-Purin-6-yl)butane-1,4-diamine (1f, C9H14N6)
Conclusion
The novel hydroxamate derivatives with the purine scaffold
were synthesized as HDACs inhibitors and evaluated. Some
White solid; yield 1.4g (70%); mp: >300°C; 1H-NMR
of the tested compounds exhibited good inhibitory activities (600MHz, D2O) δ=1.83–1.85 (m, 4H, CH2), 3.01 (t, J=6.0Hz,
against HDACs and compounds 4d, 7c′, 7f′, 7h′, 7m′, 7o′ and 2H, CH2), 3.77 (s, 2H, CH2), 8.40 (s, 1H, CH), 8.54 (s, 1H,
7p′ showed comparable inhibitory activity with SAHA and CH) ppm.
PXD101. In addition, compounds 7f′, 7l′, 7m′ were more po-
tent than positive drugs SAHA and PXD101 in cellular anti-
N1-(2-Chloro-9H-purin-6-yl)ethane-1,2-diamine (1g, C7H9ClN6)
White solid; yield 1.3g (62%); mp: 198–200°C; H-NMR
1
proliferative activity. Molecular docking study indicated that (600MHz, D2O) δ=2.51 (t, J=6.6Hz, 2H, CH2), 2.87 (d,
the conformation of 7m′ in the active site of HDAC2 was sim- J=4.8Hz, 2H, CH2), 3.53 (s, 2H, NH2), 8.13 (s, 1H, CH), 8.23
ilar to SAHA, which were oriented with the hydroxamic acid (s, 1H, NH) ppm;.
towards the catalytic center and formed metal binding with
zinc ion. Except for metal binding, the generally observed
N1-(2-Chloro-9H-purin-6-yl)propane-1,3-diamine (1j, C81H11ClN6)
White solid; yield 1.9g (82%); mp: 210–212°C; H-NMR
interations of 7m′ with HDAC2 involved hydrogen bonds and (600MHz, D2O) δ=1.96 (t, J=7.2Hz, 2H, CH2), 2.19–3.01 (m,
hydrophobic interations. Western blot analysis showed each 2H, CH2), 3.56 (s, J=6.6Hz, 2H, CH2), 7.92 (s, 1H, CH) ppm.
of the compounds, 7f′, 7l′, 7m′, 7o′ and SAHA increased his-
General Synthetic Method of Compounds 2g–2l The
tone H3 acetylation in HCT116 and K562 cell lines, and 7m′ intermediate 1g or 1j (10mmol) was dissolved in 20mL anhy-
increased the level of acetyl histone H3 in a dose-dependent drous n-butanol, then catalytic amount of TFA (1mmol) was
manner similar to SAHA.
added, and then aromatic amine (20mmol) was dropped into
the solution. The mixture was stirred for 12h at 120°C. The
resulting mixture was cooled to room temperature, filtered
Experimental
All starting materials, reagents and solvents were com- and the solid was washed with ether and ethanol to give com-
mercially available. All reagents were used without further pounds 2g–2l.
purification unless stated. IR spectra were measured on KBr
pellets on a Shimadzu FTIR-8000 spectrometer in the range C13H15N7)
of 4000–400cm−1. NMR spectra were determined on a Bruk-
White solid; yield 2.0g (75%); mp: 231–234°C; H-NMR
N6-(2-Aminoethyl)-N2-phenyl-9H-purine-2,6-diamine (2g,
1
er AV 600MHz spectrometer with D2O or dimethyl sulfoxide (600MHz, DMSO-d6) δ=3.12 (t, J=6.6Hz, 2H, CH2), 3.68
(DMSO)-d6 as the solvent, chemical shift values were reported (t, J=6.6Hz, 2H, CH2), 7.08 (s, 1H, CH), 7.28 (d, J=7.8Hz,
in parts per million (ppm) and Hertz (Hz). Mass spectra were 2H, ArH), 7.33–7.35 (m, 2H, ArH), 7.86 (s, 1H, ArH) ppm;
recorded on a AB Triple TOF 5600-1 mass spectrometer. 13C-NMR (151MHz, DMSO-d6) δ=20.30, 36.37, 115.79, 117.76,
Melting points were recorded on an electrothermal melting 119.11, 128.94, 129.85, 152.58, 158.51, 158.72ppm; high resolu-
point apparatus (WRS-1A) and uncorredted.
tion (HR)-MS-electrospray ionization (ESI): m/z=270.1389
General Synthetic Method of Compounds 1a–1g and 1j ([M+H]+, Calcd), 270.1382 (Found).
The starting purin materials (10mmol) (6-chloro-9H-purin-
N 6-(2-Aminoethyl)-N2-(4-methylphenyl)-9H-purine-2,6-
2-amine, 6-chloro-9H-purin, 2,6-dichloro-9H-purin-2-amine) diamine (2h, C14H17N7)
1
or diamines (30mmol) were dissolved in 30mL anhydrous
White solid; yield 2.2g (77%); mp: 215–217°C; H-NMR
n-butanol, triethylamine (TEA) (10mmol) was added. The (600MHz, DMSO-d6) δ=2.15 (q, J=6.6Hz, 3H, CH3), 2.12
mixture was refluxed for 6h under nitrogen atmosphere. The (t, J=6.6Hz, 2H, CH2), 3.65 (q, J=7.6Hz, 2H, CH2), 7.02
resulting mixture was cooled to room temperature, and con- (d, J=7.2Hz, 2H, ArH), 7.16 (d, J=7.8Hz, 2H, ArH), 7.90
centrated under reduced pressure. The solid was washed with (s, H, ArH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=20.30,
acetone and ethanol to give desired compounds 1a–1g, 1j.
39.10, 115.79, 117.76, 131.11, 132.31, 133.71, 152.58, 158.51,
N6-(2-Aminoethyl)-9H-purine-2,6-diamine (1a, C7H11N7)
167.49ppm; HR-MS-ESI: m/z=284.1615 ([M+H]+, Calcd),
Light yellow solid; yield 1.7g (88%); mp: 268–269°C; 284.1625 (Found) .
1H-NMR (600MHz, D2O): δ=3.02 (t, J=5.4Hz, 2H, CH2),
3.57 (t, J=6.0Hz, 2H, CH2), 7.72 (s, 1H, CH) ppm.
N 6-(2-Aminoethyl)-N2-(4-hydroxyphenyl)-9H-purine-2,6-
diamine (2i, C13H15N7O)
White solid; yield 2.1g (75%); mp: 201–204°C; H-NMR
N6-(3-Aminopropyl)-9H-purine-2,6-diamine (1b, C8H13N7)
1
1
White solid; yield 1.8g (89%); mp: 270–271°C; H-NMR (600MHz, DMSO-d6) δ=3.11 (t, J=7.2Hz, 2H, CH2), 3.64 (q,
(600MHz, D2O): δ=2.01–2.03 (m, 2H, CH2), 3.02 (t, J=7.2Hz, J=6.6Hz, 2H, CH2), 6.70 (d, J=7.8Hz, 2H, ArH), 7.11–7.13
2H, CH2), 3.62 (s, 2H, CH2), 8.02 (s, 1H, CH) ppm.
(m, 2H, ArH), 7.79 (s, H, ArH) ppm; 13C-NMR (151MHz,
N6-(4-Aminobutyl)-9H-purine-2,6-diamine (1c, C9H15N7)
DMSO-d6) δ=21.30, 39.37, 123.59, 125.11, 125.71, 128.94,
1
White solid; yield 1.7g (79%); mp: 278–279°C; H-NMR 131.85, 132.58, 145.52, 148.72ppm; HR-MS-ESI: m/z=286.1416
(600MHz, D2O): δ=1.06 (m, 4H, CH2), 3.31–3.32 (m, 4H, ([M+H]+, Calcd). Found (286.1413).
CH2), 8.01 (s, 1H, CH), 8.95 (s, 1H, NH) ppm.
N 6-(3-Aminopropyl)-N2-(4-methyl-phenyl)-9H-purine-2,6-
diamine (2j, C15H19N7)
N6-(6-Aminohexyl)-9H-purine-2,6-diamine (1d, C11H19N7)
White solid; yield 1.6g (66%); mp: >300°C; 1H-NMR
White solid; yield 2.6g (86%); mp: 245–247°C; H-NMR
1
(600MHz, D2O): δ=1.26–1.27 (m, 4H, CH2), 1.50–1.53 (m, (600MHz, DMSO-d6) δ=1.84 (t, J=6.6Hz, 3H, CH3), 2.83 (q,
4H, CH2), 2.83 (t, J=7.8Hz, 2H, CH2), 3.31 (s, 2H, CH2), 7.63 J=7.2Hz, 2H, CH2), 2.98 (t, J=7.2Hz, 2H, CH2), 3.47–3.49
(s, 1H, CH) ppm.
(m, 2H, CH2), 7.08 (d, J=7.8Hz, 2H, ArH), 7.18–7.21 (m, 2H,
N 1-(9H-Purine-6-yl)ethane-1,2-diamine (1e, C7H10N6)
ArH), 7.84 (s, H, ArH) ppm; 13C-NMR (151MHz, DMSO-
White solid; yield 1.1g (60%); mp: >300°C; 1H-NMR d6) δ=20.30, 39.80, 39.90, 117.23, 119.74, 131.15, 131.16,
(600MHz, D2O) δ=3.27 (t, J=6.0Hz, 2H, CH2), 3.84 (t, 132.24, 132.51, 150.25, 154.51, 165.43ppm; HR-MS-ESI:

Vol. 66, No. 4 (2018)
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445
m/z=298.1702 ([M+H]+, Calcd), 298.1709 (Found).
(NH) cm−1.
N 6-(3-Aminopropyl)-N2-(4-hydroxyphenyl)-9H-purine-2,6-
General Synthetic Method of Compounds 4a–4l At
diamine (2k, C14H17N7O)
room temperature, to a solution of hydroxylamine hydrochlo-
1
White solid; yield 2.6g (87%); mp: 237–239°C; H-NMR ride (5mmol) in 10mL anhydrous methanol, NaOH (10mmol)
(600MHz, DMSO-d6) δ=1.87 (t, J=6.6Hz, 2H, CH2), 2.87 was added. After stirring the mixture at room temperature
(t, J=7.2Hz, 2H, CH2), 3.51 (t, J=6.6Hz, 2H, CH2), 7.10 (d, for 30min, adding it to the previous 3, and the mixture
J=9.5Hz, 2H, ArH), 7.22 (q, J=8.4Hz, 2H, ArH), 7.77 (s, 1H, was stirred for 5h at 60°C. Then most of the methanol was
CH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=37.21, 39.59, evaporated and the residues were adjusted to pH 5–6 with
39.98, 115.39, 116.54, 116.66, 118.64, 121.89, 124.13, 157.00, HCl (1mol/L). The solution was concentrated under reduced
157.04ppm; HR-MS-ESI: m/z=300.1495 ([M+H]+, Calcd), pressure and the crude product was purified by chromatogra-
300.1488 (Found).
N 6-(3-Aminopropyl)-N2-(4-methoxyphenyl)-9H-purine-2,6- give desired compounds 4a–4l.
diamine (2l, C15H19N7O)
1-(2-(2-Amino-9H-purin-6-yl)amino)ethyl)-3-hydroxyurea
White solid; yield 2.4g (76%); mp: 199–202°C; H-NMR (4a, C8H12N8O2)
phy on a silica gel column (methanol/dichloromethane, 1:3) to
1
(600MHz, D2O) δ=1.90 (t, J=6.6Hz, 2H, CH2), 1.96–1.99 (m,
Isolated yield: 42%; white powder; mp: 185–187°C; IR
2H, CH2), 2.90 (t, J=7.2Hz, 2H, CH2), 3.53 (s, 2H, NH2), 3.75 (KBr): ν=1603 (CO-acetyl), 2949 (CH-aliph), 3070 (CH-aryl),
(s, 3H, CH3), 6.90 (d, J=9.0Hz, 2H, ArH),7.30 (d, J=9.0Hz, 3200–3500 (br NH2, NH, OH) cm−1; 1H-NMR (600MHz,
2H, ArH), 7.84 (s, 1H, CH) ppm; 13C-NMR (151MHz, DMSO- DMSO-d6) δ=3.13 (t, J=5.4Hz, 2H, CH2), 3.16 (t, J=5.4Hz,
d6) δ=15.05, 19.80, 20.23, 35.94, 118.92, 119.76, 128.11, 128.58, 2H, CH2), 4.16 (s, 2H, NH2), 6.82 (s, 2H, NH), 8.31 (s, 1H,
129.41, 139.23, 141.75, 156.22, 161.51,178.66ppm; HR-MS-ESI: CH), 8.61 (s, 1H, NH) ppm; 13C-NMR (151MHz, DMSO-d6)
m/z=314.1651 ([M+H]+, Calcd), 314.1665 (Found).
δ=27.9, 56.5, 86.8, 101.9, 116.8, 139.0, 147.0, 154.7, 162.0ppm;
General Synthetic Method of Compounds 3a–3l Com- HR-MS-ESI: m/z=253.1161 ([M+H]+, Calcd), 253.1164
pounds 1a–1f or 2g–2l (5mmol) was dissolved by 10cm3 (Found).
H2O and 15cm3 acetonitrile, respectively, and then NaHCO3
1-(3-(2-Amino-9H-purin-6-yl)amino)propyl)-3-hydroxyurea
(5mmol) was added, the solution was stirred at 0°C, 4-nitro- (4b, C9H14N8O2)
phenyl chloroformate (5mmol) pre-dissolved with acetonitrile
Isolated yield: 52%; white powder; mp: 190–193°C; IR
was dropped into the solution. The mixture was stirred for (KBr): ν=1601 (CO-acetyl), 2908 (CH-aliph), 3057 (CH-aryl),
0.5–1h at 0°C. Because of aftertreatments of 3a–3f were 3200–3500 (br. NH2, NH, OH) cm−1; 1H-NMR (600MHz,
difficult, they were concentrated and then directly used for DMSO-d6) δ=1.63 (t, J=5.4Hz, 2H, CH2), 2.81 (t, J=6.6Hz,
synthesizing 4a–4l without further purification. The result- 2H, CH2), 3.09 (t, J=6.0Hz, 2H, CH2), 5.66 (s, 2H, NH2), 7.08
ing precipitations of 3g–3l were filtered and then washed with (s, 1H, NH), 7.21 (s, 1H, NH), 7.63 (s, 1H, NH), 8.31 (s, 1H,
acetonitrile to give compounds 3g–3l.
4-Nitrophenyl(2-(2-(phenylamino)-9H-purin-6-yl)amino)ethyl)- δ=26.5, 26.6, 73.2, 90.5, 131.1, 136.4, 148.6, 156.7, 162.0ppm;
carbamate (3g, C20H18N8O4)
HR-MS-ESI: m/z=267.1318 ([M+H]+, Calcd), 267.1312
White solid; yield 1.6g (73%); mp: 214–216°C; IR (KBr): (Found).
CH), 8.71 (s, 1H, NH) ppm; 13C-NMR (151MHz, DMSO-d6)
ν=1658 (CO-acetyl), 2901 (CH-aliph), 3009 (CH-aryl), 3424
1-(4-(2-Amino-9H-purin-6-yl)amino)butyl)-3-hydroxyurea
(4c, C10H16N8O2)
(NH) cm−1.
4-Nitrophenyl(2-(2-(4-methylphenyl)amino)-9H-purin-6-
yl)amino)ethyl) carbamate (3h, C21H20N8O4)
Isolated yield: 51%; white powder; mp: 195–198°C; IR
(KBr): ν=1614 (CO-acetyl), 2845 (CH-aliph), 3097 (CH-aryl),
White solid; yield 1.5g (68%); mp: 225–226°C; IR (KBr): 3200–3500 (br. NH2, NH, OH) cm−1; 1H-NMR (600MHz,
ν=1648 (CO-acetyl), 2941 (CH-aliph), 3029 (CH-aryl), 3404 DMSO-d6) δ=1.48–1.56 (m, 4H, CH2), 3.07 (t, J=6.0Hz, 2H,
(NH) cm−1.
CH2), 3.09 (s, 2H, CH2), 4.01 (s, 2H, NH2), 6.70 (s, 2H, NH),
4-Nitrophenyl(2-(2-((4-hydroxyphenyl)amino)-9H-purin-6- 8.21 (s, 1H, CH), 8.51 (s, 1H, NH) ppm; 13C-NMR (151MHz,
yl)amino)ethyl)carbamate (3i, C20H18N8O5)
DMSO-d6) δ=26.1, 27.6, 29.8, 118.8, 41.7, 149.9, 153.6, 156.0,
White solid; yield 1.7g (75%); mp: 225–228°C; IR (KBr): 168.6ppm; HR-MS-ESI: m/z=281.1474 ([M+H]+, Calcd),
ν=1685 (CO-acetyl), 2981 (CH-aliph), 3049 (CH-aryl), 3435 281.1463 (Found).
(NH) cm−1.
1-(6-(2-Amino-9H-purin-6-yl)amino)hexyl)-3-hydroxyurea
4-Nitrophenyl(3-(2-(4-methylphenyl)amino)-9H-purin-6-yl)- (4d, C12H20N8O2)
amino)propyl)carbamate (3j, C22H22N8O4)
Isolated yield: 37%; white powder; mp: 170–172°C; IR
White solid; yield 1.5g (64%); mp: 187–190°C; IR (KBr): (KBr): ν=1620 (CO-acetyl), 2943 (CH-aliph), 3057 (CH-aryl),
ν=1645 (CO-acetyl), 2981 (CH-aliph), 3049 (CH-aryl), 3415 3200–3500 (br. NH2, NH, OH) cm−1; 1H-NMR (600MHz,
(NH) cm−1.
DMSO-d6) δ=1.27–1.33 (m, 4H, CH2), 1.42 (t, J=7.2Hz, 2H,
4-Nitrophenyl(3-(2-((4-hydroxyphenyl)amino)-9H-purin-6- CH2), 1.58 (t, J=7.2Hz, 2H, CH2), 3.04 (q, J=6.6Hz, 2H,
yl)amino)propyl)carbamate (3k, C21H20N8O5) CH2), 3.32 (s, 2H, CH2), 3.44 (s, 2H, NH2), 6.65 (t, J=6.0Hz,
White solid; yield 1.2g (72%); mp: 198–199°C; IR (KBr): 2H, NH), 7.89 (s, 1H, NH), 8.20 (s, 1H, CH), 8.50 (s, 1H,
ν=1635 (CO-acetyl), 2881 (CH-aliph), 3002 (CH-aryl), 3359 NH), 12.66 (s, 1H, OH) ppm; 13C-NMR (151MHz, DMSO-d6)
(NH) cm−1.
δ=26.0, 27.7, 29.8, 118.9, 141.7, 150.0, 153.7, 156.0, 168.6ppm;
4-Nitrophenyl(3-(2-((4-methoxyphenyl)amino)-9H-purin-6- HR-MS-ESI: m/z=309.1787 ([M+H]+, Calcd), 309.1773
yl)amino)propyl)carbamate (3l, C22H22N8O5)
White solid; yield 1.6g (65%); mp: 192–194°C; IR (KBr):
ν=1675 (CO-acetyl), 2920 (CH-aliph), 3007 (CH-aryl), 3410
(Found).
1-(2-(9H-Purin-6-yl)amino)ethyl)-3-hydroxyurea (4e, C8H11N7O2)
Isolated yield: 50%; white powder; mp: 185–186°C; IR

446
Chem. Pharm. Bull.
Vol. 66, No. 4 (2018)
(KBr): ν=1612 (CO-acetyl), 2999 (CH-aliph), 3057 (CH-aryl), (d, J=9.6Hz, 2H, NH), 6.82–6.85 (m, 2H, ArH), 7.22 (t,
1
3255 (NH), 3443 (OH) cm−1; H-NMR (600MHz, DMSO-d6) J=7.2Hz, 2H, ArH), 7.71 (s, 1H, NH), 7.73 (s, 1H, NH), 7.83
δ=3.35 (t, J=7.2Hz,2H, CH2), 3.73 (q, J=7.8Hz, 2H, CH2), (s, 1H, CH), 8.30 (s, 1H, NH) ppm; 13C-NMR (151MHz,
7.60 (s, 1H, CH), 8.90 (s, 1H, CH), 9.86 (s, 1H, NH) ppm; DMSO-d6) δ=20.23, 64.78, 79.18, 118.92, 119.76, 128.11,
13C-NMR (151MHz, DMSO-d6) δ=25.4, 31.2, 42.9, 49.1, 115.4, 129.41, 141.75, 156.21, 161.51, 178.66ppm; HR-MS-ESI:
119.9, 123.5, 131.5, 141.7, 149.9, 152.9, 153.7, 174.1ppm; HR- m/z=357.1787 ([M+H]+, Calcd), 357.1785 (Found).
MS-ESI: m/z=238.1052 ([M+H]+, Calcd), 238.1041 (Found).
1-(3-(2-(4-Hydroxyphenyl)amino)-9H-purin-6-yl)amino)-
1-(4-(9H-Purin-6-yl)amino)butyl)-3-hydroxyurea (4f, C10H15N7O2) propyl)-3-hydroxyurea (4k, C15H18N8O3)
Isolated yield: 45%; white powder; mp: 195–196°C; IR
Isolated yield: 25%; white powder; mp: 184–187°C; IR
(KBr): ν=1688 (CO-acetyl), 2979 (CH-aliph), 3255 (NH), 3413 (KBr): ν=1624 (CO-acetyl), 2901 (CH-aliph), 3022 (CH-aryl),
1
1
(OH) cm−1; H-NMR (600MHz, DMSO-d6) δ=1.45–1.48 (m, 3220 (NH), 3438 (OH) cm−1; H-NMR (600MHz, DMSO-
2H, CH2), 1.58 (t, J=7.2Hz, 2H, CH2), 3.04 (q, J=7.2Hz, 2H, d6) δ=1.09 (t, J=6.6Hz, 1H, CH2), 1.67–1.72 (m, 2H, CH2),
CH2), 3.46 (q, J=7.2Hz, 2H, CH2), 6.73 (t, J=6.0Hz, 1H, NH), 3.27–3.30 (m, 2H, CH2), 3.51 (s, 1H, OH), 5.90 (s, 2H, NH),
7.65 (s, 1H, CH), 8.09 (s, 1H, CH), 8.12 (t, J=9.6Hz, 1H, NH), 6.67 (q, J=8.4Hz, 2H, ArH), 7.54 (t, J=6.0Hz, 2H, ArH), 7.71
8.24 (s, 1H, NH), 8.54 (t, J=5.4Hz, 1H, NH), 12.89 (s, 1H, (s, 1H, NH), 7.73 (s, 1H, NH), 8.32 (s,1H, NH) ppm; 13C-NMR
OH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=24.9, 26.3, 28.6, (151MHz, DMSO-d6) δ=21.31, 27.56, 36.57, 39.59, 115.22,
28.9, 29.7, 34.2, 43.7, 49.1, 115.5, 118.9, 124.0, 130.6, 142.3, 119.17, 120.61, 127.94, 128.32, 180.77ppm; HR-MS-ESI:
151.0, 153.2, 154.4, 175.0ppm; HR-MS-ESI: m/z=266.1365 m/z=359.1580 ([M+H]+, Calcd). Found (359.1585).
([M+H]+, Calcd), 266.1378 (Found).
1-(2-(2-(Phenylamino)-9H-purin-6-yl)amino)ethyl)-3- propyl)-3-hydroxyurea (4l, C16H20N8O3)
1-(3-(2-(4-Methoxyphenyl)amino)-9H-purin-6-yl)amino)-
hydroxyurea (4g, C14H16N8O2)
Isolated yield: 36%; white powder; mp: 196–199°C; IR
Isolated yield: 32%; white powder; mp: 179–182°C; IR (KBr): ν=1620 (CO-acetyl), 2935 (CH-aliph), 3366 (NH)
(KBr): ν=1610 (CO-acetyl), 2855 (CH-aliph), 3000–3400 (br. cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.71 (t, J=6.6Hz,
1
CH-aryl, NH, OH) cm−1; H-NMR (600MHz, D2O) δ=3.16 (t, 2H, CH2), 2.39 (s, 3H, CH3), 3.15 (t, J=6.6Hz, 2H, CH2), 3.49
J=6.6Hz, 2H, CH2), 3.37 (t, J=6.6Hz, 2H, CH2), 4.18 (s, 1H, (t, J=6.6Hz, 2H, CH2), 6.97 (s, 1H, NH), 7.03 (d, J=8.4Hz,
NH), 6.82 (s, 1H, NH), 7.20 (s, 2H, ArH), 7.30 (d, J=7.6Hz, 2H, ArH), 7.51 (s, 1H, NH), 7.69 (d, J=7.8Hz, 2H, ArH), 7.76
1H, ArH), 7.82 (d, J=7.1Hz, 1H, ArH), 8.00 (s, 1H, CH) (s, 1H, NH), 8.32 (s, 1H, NH), 8.62 (s, 1H, CH), 8.67 (s, 1H,
ppm;13C-NMR (151MHz, DMSO-d6) δ=21.32, 39.59, 76.10, NH), 12.42 (s, 1H, OH) ppm; 13C-NMR (151MHz, DMSO-
115.79, 119.76, 129.14, 129.28, 129.65, 129.84, 131.16, 137.99, d6) δ=20.30, 36.39, 39.90, 119.17, 128.83, 152.58, 158.51,
139.05ppm; HR-MS-ESI: m/z=329.1474 ([M+H]+, Calcd), 158.72ppm; HR-MS-ESI: m/z=395.1556 ([M+Na]+, Calcd),
329.1478 (Found).
395.1545 (Found).
1-(2-(2-(4-Methyl-phenyl)amino)-9H-purin-6-yl)amino)-
ethyl)-3-hydroxyurea (4h, C15H18N8O2)
General Synthetic Method of Compounds 5b′–5i′ Com-
pounds 5b′–i′ could be obtained by reaction of 2,6-dichlo-
Isolated yield: 26%; white powder; mp: 207–210°C; IR ro-9H-purine and different aliphatic amines and aromatic
(KBr): ν=1639 (CO-acetyl), 2918 (CH-aliph), 3100 (CH-aryl), amines, their synthesis methods were same as that of 1a–1d.
3307 (NH), 3398 (OH) cm−1; 1H-NMR (600MHz, D2O)
2-Chloro-6-methylamino-9H-purine (5b′, C6H6ClN5)
δ=2.24 (q, J=6.6Hz, 3H, CH3), 3.57 (t, J=6.8Hz, 2H, CH2),
White solid; yield 1.6g (89%); mp: 275–276°C; IR (KBr):
4.16 (s, 2H, CH2), 6.22 (s, 2H, NH), 7.05 (d, J=7.8Hz, 2H, ν=1651 (NH), 2831 (CH-aliph), 3419 (NH) cm−1.
ArH), 7.69 (s, 2H, ArH), 8.07 (s, H, CH), 8.97 (s, H, NH),
2-Chloro-6-ethylamino-9H-purin (5c′, C7H8ClN5)
White solid; yield 1.8g (92%); mp: 279–280°C; IR (KBr):
11.90 (s, H, OH) ppm; 13C-NMR (151MHz, DMSO-d6)
δ=20.30, 36.37, 39.03, 114.33, 119.19, 121.19, 122.27, 127.96, ν=1651 (NH), 2877 (CH-aliph), 3419 (NH) cm−1.
129.65, 129.79, 133.16, 155.72ppm; HR-MS-ESI: m/z=343.1631
2-Chloro-6-propylamino-9H-purin (5d′, C8H10ClN5)
White solid; yield 1.8g (85%); mp: >300°C; IR (KBr):
([M+H]+, Calcd), 343.1626 (Found).
1-(2-(2-(4-Hydroxyphenyl)amino)-9H-purin-6-yl)amino)- ν=1651 (NH), 2877 (CH-aliph), 3419 (NH) cm−1.
ethyl)-3-hydroxyurea (4i, C14H16N8O3)
2-Chloro-6-phenylamino-9H-purin (5e′, C11H8ClN5)
Isolated yield: 52%; white powder; mp: 178–180°C; IR
White solid; yield 1.9g (76%); mp: 297–298°C; IR (KBr):
(KBr): ν=1581 (CO-acetyl), 2939 (CH-aliph), 3000–3400 (br. ν=1651 (NH), 2881 (CH-aliph), 3081 (CH-aryl), 3411 (NH)
CH-aryl, NH, OH) cm−1; 1H-NMR (600MHz, DMSO-d6) cm−1.
δ=3.55 (t, J=6.6Hz, 2H, CH2), 4.13 (t, J=5.6Hz, 2H, CH2),
6.67 (d, J=7.8Hz, 2H, ArH), 7.53–7.56 (m, 2H, ArH), 8.07 (s,
2-Chloro-6-(4-methylphenyl)amino-9H-purin (5f′, C12H10ClN5)
White solid; yield 1.9g (75%); mp: >300°C; IR (KBr):
1H, CH), 8.75 (s, 1H, NH), 8.88 (s, 1H, NH), 11.86 (s, 1H, OH) ν=1651 (NH), 2877 (CH-aliph), 3197 (CH-aryl), 3415 (NH)
ppm; 13C-NMR (151MHz, DMSO-d6) δ=21.32, 76.10, 129.28, cm−1.
129.65, 129.84, 131.16, 139.07, 153.57, 154.21, 168.50ppm; HR-
2-Chloro-6-(4-methoxyphenyl)amino-9H-purin (5g′, C12H10ClN5O)
White solid; yield 2.2g (81%); mp: >300°C; IR (KBr):
MS-ESI: m/z=343.1267 ([M-H]-, Calcd), 343.1261 (Found).
1-(3-(2-(4-Methyl-phenyl)amino)-9H-purin-6-yl)amino)- ν=1666 (NH), 2864 (CH-aliph), 3074 (CH-aryl), 3435 (NH)
propyl)-3-hydroxyurea (4j, C16H20N8O2)
cm−1.
2-Chloro-6-(4-chlorophenyl)amino-9H-purin (5h′, C11H7Cl2N5)
White solid; yield 1.9g (69%); mp: >300°C; IR (KBr):
Isolated yield: 20%; white powder; mp: 220–223°C; IR
(KBr): ν=1627 (CO-acetyl), 2922 (CH-aliph), 3061 (CH-
aryl), 3200–3400 (br NH, OH) cm−1; 1H-NMR (600MHz, ν=1643 (NH), 2937 (CH-aliph), 3217 (NH) cm−1.
DMSO-d6) δ=1.09 (t, J=6.0Hz, 2H, CH2), 1.72 (s, 3H, CH3),
3.16 (t, J=6.0Hz, 2H, CH2), 3.30–3.33 (m, 2H, CH2), 5.93
2-Chloro-6-(4-hydroxyphenyl)amino-9H-purin (5i′, C11H8ClN5O)
White solid; yield 1.9g (72%); mp: >300°C; IR (KBr):

Vol. 66, No. 4 (2018)
Chem. Pharm. Bull.
447
ν=2879 (CH-aliph), 3062 (CH-aryl), 3200–3400 (br NH, OH) J=7.2Hz, 3H, CH3), 2.07 (q, J=7.2Hz, 2H, CH2), 2.26 (s, 3H,
cm−1.
CH3), 2.31 (t, J=7.2Hz, 2H, CH2), 3.97 (m, 2H, CH2), 4.18
General Synthetic Method of Compounds 6a′–6p′ (t, J=6.6Hz, 2H, CH2), 7.14 (t, J=7.8Hz, 2H, ArH), 7.69 (d,
2,6-dichloro-9H-purine (5mmol) or intermediates 5 was dis- J=8.4Hz, 2H, ArH), 8.27 (s, 1H, CH), 10.18 (s, 1H, NH) ppm.
solved in 20mL anhydrous DMF, then K2CO3 (8mmol) was
Ethyl-4-(2-chloro-6-(4-methoxyphenyl)amino-9H-purin-9-
added, and ethyl 4-bromobutanoate or ethyl 8-bromooctanoate yl)butanoate (6g′, C18H20ClN5O3)
(8mmol) was dropped into the solution. Cut off from the air,
Isolated yield: 62%; light yellow soild; mp: 90–92°C; IR
the solution was stirred at room temperature for overnight, (KBr): ν=1577 (CO-acetyl), 2980 (CH-aliph), 3071 (CH-aryl),
and then adjusted to pH 7–8 with HCl (1mol/L), then ex- 3201 (NH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.13
tracted with 50mL ×3 ethyl acetate and the organic phase (t, J=7.2Hz, 3H, CH3), 2.08 (t, J=7.2Hz, 2H, CH2), 2.32 (t,
was combined, and washed with brine, dried over MgSO4 and J=7.2Hz, 2H, CH2), 2.88 (s, 2H, CH2), 3.75 (s, 3H, CH3), 4.18
evaporated. The crude product was purified by chromatogra- (t, J=6.6Hz, 2H, CH2), 6.94 (d, J=2.4Hz, 2H, ArH), 7.69 (d,
phy on a silica gel column (EtOAc/petroleum ether, 1:1) to J=9.0Hz, 2H, ArH), 8.28 (s, 1H, CH), 10.13 (s, 1H, NH) ppm.
give desired compounds 6a′–6p′.
Ethyl-4-(2-chloro-6-(4-chlorophenyl)amino-9H-purin-9-
Ethyl-4-(2,6-dichloro-9H-purin-9-yl)butanoate (6a′, C11H12Cl2N4O2) yl)butanoate (6h′, C17H17Cl2N5O2)
Isolated yield: 82%; light yellow oil; IR (KBr): ν=1541
Isolated yield: 77%; colorless oil; IR (KBr): ν=1633 (CO-
(CO-acetyl), 2935 (CH-aliph), 3338 (NH) cm−1; 1H-NMR acetyl), 2937 (CH-aliph), 3081 (CH-aryl), 3417 (NH) cm−1;
(600MHz, DMSO-d6) δ=1.15 (t, J=7.2Hz, 3H, CH3), 2.04 1H-NMR (600MHz, DMSO-d6) δ=1.13 (t, J=7.2Hz, 3H, CH3),
(t, J=7.2Hz, 2H, CH2), 2.30 (t, J=7.2Hz, 2H, CH2), 2.50 (q, 2.08 (t, J=7.2Hz, 2H, CH2), 2.33 (t, J=7.2Hz, 2H, CH2), 3.98
J=7.8Hz, 2H, CH2), 4.14 (t, J=7.2Hz, 2H, CH2), 8.13 (s, 1H, (q, J=7.2Hz, 2H, CH2), 4.20 (t, J=6.6Hz, 2H, CH2), 7.40 (d,
CH) ppm.
J=8.4Hz, 2H, ArH), 7.89 (d, J=9.0Hz, 2H, ArH), 8.32 (s, 1H,
Ethyl-4-(2-chloro-6-methylamino-9H-purin-9-yl)butanoate CH), 10.41 (s, 1H, NH) ppm.
(6b′, C12H16ClN5O2)
Ethyl-4-(2-chloro-6-(4-hydroxyphenyl)amino-9H-purin-9-
Isolated yield: 83%; light yellow oil; IR (KBr): ν=1643 yl)butanoate (6i′, C17H18ClN5O3)
(CO-acetyl), 2937 (CH-aliph), 3217 (NH) cm−1; 1H-NMR
Isolated yield: 77%; light yellow soild; mp: 162–164°C;
(600MHz, DMSO-d6) δ=1.15 (t, J=7.2Hz, 3H, CH3), 2.04 IR (KBr): ν=786, 825, 1031, 1178, 1238, 1429, 1500, 1620,
1
(t, J=7.2Hz, 2H, CH2), 2.29 (t, J=7.2Hz, 2H, CH2), 2.92 (d, 2935, 3365cm−1; H-NMR (600MHz, DMSO-d6) δ=1.17 (t,
J=4.2Hz, 3H, CH3), 3.99 (q, J=7.2Hz, 2H, CH2), 4.14 (t, J=7.2Hz, 3H, CH3), 1.79 (t, J=7.2Hz, 2H, CH2), 2.25 (t,
J=6.6Hz, 2H, CH2), 8.12 (s, 1H, CH), 8.15 (s, 1H, NH) ppm.
Ethyl-4-(2-chloro-6-ethylamino-9H-purin-9-yl)butanoate J=7.2Hz, 2H, CH2), 6.75 (d, J=9.0Hz, 2H, ArH), 7.52 (d,
(6c′, C13H18ClN5O2) J=8.4Hz, 2H, ArH), 8.27 (s, 1H, CH), 9.29 (s, 1H, NH), 10.00
J=7.2Hz, 2H, CH2), 4.03 (t, J=7.2Hz, 2H, CH2), 4.13 (t,
Isolated yield: 79%; colorless oil; IR (KBr): ν=1620 (s, 1H, OH) ppm.
(CO-acetyl), 2943 (CH-aliph), 3348 (NH) cm−1; 1H-NMR
Ethyl-8-(2,6-dichloro-9H-purin-9-yl)octanoate (6j′, C15H20Cl2N4O2)
(600MHz, DMSO-d6) δ=1.12–1.17 (m, 6H, CH3), 2.05 (q,
Isolated yield: 79%; light yellow oil; IR (KBr): ν=1637
J=6.6Hz, 2H, CH2), 2.29 (t, J=7.2Hz, 2H, CH2), 3.44 (CO-acetyl), 2943 (CH-aliph) cm−1; 1H-NMR (600MHz,
(t, J=6.0Hz, 2H, CH2), 3.96–4.00 (m, 2H, CH2), 4.14 (t, DMSO-d6) δ=1.21 (t, J=5.4Hz, 3H, CH3), 1.26–1.27 (m, 6H,
J=7.2Hz, 2H, CH2), 8.11 (s, 1H, CH), 8.21 (s, 1H, NH) ppm.
CH2), 1.47 (t, J=7.2Hz, 2H, CH2), 1.80–1.85 (m, 2H, CH2),
Ethyl-4-(2-chloro-6-propylamino-9H-purin-9-yl)butanoate 2.21 (t, J=7.2Hz, 2H, CH2), 3.99 (m, 2H, CH2), 4.23 (m, 2H,
(6d′, C14H20ClN5O2)
CH2), 8.10 (s, 1H, CH) ppm.
Isolated yield: 71%; colorless oil; IR (KBr): ν=1651
Ethyl-8-(2-chloro-6-propylamino-9H-purin-9-yl)octanoate
(CO-acetyl), 2877 (CH-aliph), 3200 (NH) cm−1; 1H-NMR (6k′, C18H28ClN5O2)
(600MHz, DMSO-d6) δ=0.87 (t, J=7.2Hz,3H, CH3), 1.15
Isolated yield: 71%; colorless oil; IR (KBr): ν=1525
(t, J=7.2Hz, 3H, CH3), 1.60 (t, J=7.2Hz, 2H, CH2), 2.06 (t, (CO-acetyl), 2947 (CH-aliph), 3336 (NH) cm−1; 1H-NMR
J=7.2Hz, 2H, CH2), 2.31 (t, J=7.2Hz, 2H, CH2), 3.35–3.40 (600MHz, DMSO-d6) δ=0.94 (t, J=6.0Hz, 3H, CH3),
(m, 2H, CH2), 4.02 (t, J=7.2Hz, 2H, CH2), 4.15 (t, J=7.2Hz, 1.23–1.28 (m, 5H, CH2, CH3), 1.49 (t, J=7.2Hz, 2H, CH2),
2H, CH2), 8.13 (s, 1H, CH), 8.27 (s, 1H, NH) ppm.
Ethyl-4-(2-chloro-6-phenylamino-9H-purin-9-yl)butanoate (t, J=7.8Hz, 2H, CH2), 2.74 (t, J=7.8Hz, 2H, CH2), 2.90 (s,
(6e′, C17H18ClN5O2) 2H, CH2), 3.38 (t, J=6.6Hz, 2H, CH2), 4.04 (t, J=7.2Hz, 2H,
1.60 (q, J=7.2Hz, 2H, CH2), 1.77 (t, J=7.2Hz, 2H, CH2), 2.25
Isolated yield: 69%; white soild; mp: 133–134°C; IR CH2), 4.09 (t, J=6.6Hz, 2H, CH2), 8.16 (s, 1H, CH), 8.26 (t,
(KBr): ν=1651 (CO-acetyl), 2877 (CH-aliph), 3060 (CH-aryl), J=5.4Hz, 1H, NH) ppm.
1
3419 (NH) cm−1; H-NMR (600MHz, DMSO-d6) δ=1.15 (t,
Ethyl-8-(2-chloro-6-phenylamino-9H-purin-9-yl)octanoate
J=7.2Hz, 3H, CH3), 2.08 (q, J=5.4Hz, 2H, CH2), 2.34 (t, (6l′, C21H26ClN5O2)
J=7.2Hz, 2H, CH2), 4.00 (t, J=7.2Hz, 2H, CH2), 4.20 (t,
Isolated yield: 78%; light yellow oil; IR (KBr): ν=1625
J=7.2Hz, 2H, CH2), 7.09 (q, J=7.2Hz, 1H, ArH), 7.36 (q, (CO-acetyl), 2941 (CH-aliph), 3087 (CH-aryl), 3224 (NH)
J=7.2Hz, 2H, ArH), 7.84 (q, J=7.8Hz, 2H, ArH), 8.31 (s, 1H, cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.22 (t, J=7.2Hz,
CH), 10.25 (s, 1H, NH) ppm.
Ethyl-4-(2-chloro-6-(4-methylphenyl)amino-9H-purin-9- CH2), 1.79 (t, J=7.2Hz, 2H, CH2), 2.23 (t, J=7.2Hz, 2H, CH2),
yl)butanoate (6f′, C18H20ClN5O2) 4.01 (t, J=7.2Hz, 2H, CH2), 4.13 (t, J=7.2Hz, 2H, CH2), 7.08
3H, CH3), 1.24–1.28 (m, 6H, CH2), 1.47 (t, J=7.2Hz, 2H,
Isolated yield: 70%; white soild; mp: 177–180°C; IR (KBr): (s, 1H, ArH), 7.36 (q, J=7.2Hz, 2H, ArH), 7.86 (d, J=7.2Hz,
ν=1600 (CO-acetyl), 2970 (CH-aliph), 3058 (CH-aryl), 3458 2H, ArH), 8.33 (s, 1H, CH), 10.28 (s, 1H, NH) ppm.
(NH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.12 (t,

448
Chem. Pharm. Bull.
Vol. 66, No. 4 (2018)
Ethyl-8-(2-chloro-6-(4-methylphenyl)amino-9H-purin-9- m/z=288.0055 ([M−H]−, Calcd). Found (288.0066).
yl)octanoate (6m′, C22H28ClN5O2) 4-(2-Chloro-6-methylamino-9H-purin-9-yl)-N-hydroxybuta-
Isolated yield: 61%; white soild; mp: 140–142°C; IR (KBr): namide (7b′, C10H13ClN6O2)
ν=1593 (CO-acetyl), 2972 (CH-aliph), 3020 (CH-aryl), 3317
Isolated yield: 53%; white powder; mp: 112–113°C; IR
(NH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.28 (t, (KBr): ν=1653 (CO-acetyl), 2880 (CH-aliph), 3055 (CH-aryl),
J=6.6Hz, 3H, CH3), 1.47 (m, 6H, CH2), 1.50 (t, J=7.2Hz, 3306 (NH), 3400 (OH) cm−1; H-NMR (600MHz, DMSO-d6)
1
2H, CH2), 1.80 (m, 2H, CH2), 2.25 (m, 2H, CH2), 2.29 (s, δ=1.94–2.03 (m, 4H, CH2), 2.92 (d, J=6.0Hz, 3H, CH3), 4.10
3H, CH3), 4.03 (t, J=7.2Hz, 2H, CH2), 4.14 (q, J=7.2Hz, 2H, (t, J=9.0Hz, 2H, CH2), 8.13 (s, H, CH), 8.16 (s, H, NH), 8.71
CH2), 7.17 (d, J=8.4Hz, 2H, ArH), 7.71 (d, J=8.4Hz, 2H, (s, 1H, NH), 10.37 (s, 1H, NH) ppm; 13C-NMR (151MHz,
ArH), 8.32 (s, 1H, CH), 10.20 (s, 1H, ArH) ppm.
DMSO-d6) δ: 26.1, 27.7, 29.8, 43.3, 118.8, 141.7, 150.0, 153.6,
Ethyl-8-(2-chloro-6-(4-methoxyphenyl)amino-9H-purin-9- 156.0, 168.6ppm; HR-MS-ESI: m/z=283.0710 ([M−H]−,
yl)octanoate (6n′, C22H28ClN5O3)
Calcd), 283.0718 (Found).
Isolated yield: 60%; white soild; mp: 152–154°C; IR
4-(2-Chloro-6-ethylamino-9H-purin-9-yl)-N-hydroxybu-
(KBr): ν=1596 (CO-acetyl), 2976 (CH-aliph), 3010 (CH-aryl), tanamide (7c′, C11H15ClN6O2)
1
3456 (NH) cm−1; H-NMR (600MHz, DMSO-d6) δ=1.16 (t,
Isolated yield: 51%; white powder; mp: 117–118°C; IR
J=7.2Hz, 3H, CH3), 1.23–1.29 (m, 6H, CH2), 1.46 (t, J=7.2Hz, (KBr): ν=1666 (CO-acetyl), 2864 (CH-aliph), 3074 (CH-aryl),
1
2H, CH2), 2.74 (t, J=7.2Hz, 2H, CH2), 2.90 (s, 2H, CH2), 3.76 3180 (NH), 3435 (OH) cm−1; H-NMR (600MHz, DMSO-d6)
(s, 3H, CH3), 4.04 (t, J=6.0Hz, 2H, CH2), 4.14 (t, J=6.6Hz, δ=1.17 (t, J=9.0Hz, 3H, CH3), 2.01 (t, J=6.0Hz, 2H, CH2),
2H, CH2), 6.95 (q, J=8.9Hz, 2H, ArH), 7.70 (d, J=7.9Hz, 2H, 2.23 (t, J=9.0Hz, 2H, CH2), 3.45 (t, J=6.0Hz, 2H, CH2),
ArH), 8.30 (s, 1H, CH), 10.14 (s, 1H, NH) ppm.
4.12 (t, J=9.0Hz, 2H, CH2), 8.13 (s, 1H, CH), 8.25 (s, 1H,
Ethyl-8-(2-chloro-6-(4-chlorophenyl)amino-9H-purin-9- NH), 12.16 (s, 1H, OH) ppm; 13C-NMR (151MHz, DMSO-
yl)octanoate (6o′, C21H25Cl2N5O2)
d6) δ=14.9, 25.2, 30.8, 35.4, 51.8, 118.6, 141.6, 150.2, 153.7,
Isolated yield: 71%; white soild;, mp: 258–260°C; IR 155.4, 173.1ppm; HR-MS-ESI: m/z=298.0945 ([M]+, Calcd),
(KBr): ν=1637 (CO-acetyl), 2947 (CH-aliph), 3061 (CH-aryl), 298.0933 (Found).
1
3413 (NH) cm−1; H-NMR (600MHz, DMSO-d6) δ=1.17 (t,
4-(2-Chloro-6-propylamino-9H-purin-9-yl)-N-hydroxybu-
J=7.2Hz, 3H, CH3), 1.24–1.25 (m, 6H, CH2), 1.49 (t, J=7.2Hz, tanamide (7d′, C12H17ClN6O2)
2H, CH2), 1.82 (t, J=7.2Hz, 2H, CH2), 2.25 (t, J=7.8Hz, 2H,
Isolated yield: 46%; white powder; mp: 194–195°C; IR
CH2), 4.04 (q, J=7.2Hz, 2H, CH2), 4.17 (t, J=6.6Hz, 2H, (KBr): ν=1606 (CO-acetyl), 2912 (CH-aliph), 3305 (NH),
1
CH2), 7.42 (d, J=9.2Hz, 2H, ArH), 7.92 (d, J=8.0Hz, 2H, 3400 (OH) cm−1; H-NMR (600MHz, DMSO-d6) δ=0.89 (t,
ArH), 8.37 (s, 1H, CH), 10.44 (s, 1H, NH) ppm.
J=9.0Hz, 3H, CH3), 1.60 (q, J=6.0Hz, 2H, CH2), 1.99–2.03
Ethyl-8-(2-chloro-6-(4-hydroxyphenyl)amino-9H-purin-9- (m, 2H, CH2), 2.23 (t, J=9.0Hz, 2H, CH2), 3.37–3.39 (m,
yl)octanoate (6p′, C21H26ClN5O3)
2H, CH2), 4.12 (t, J=6.0Hz, 2H, CH2), 8.14 (s, 1H, CH), 8.27
Isolated yield: 71%; white soild; mp: 158–160°C; IR (s, 1H, NH), 12.17 (s, 1H, OH) ppm; 13C-NMR (151MHz,
(KBr): ν=1633 (CO-acetyl), 2837 (CH-aliph), 3090 (CH-aryl), DMSO-d6) δ=11.8, 22.6, 25.3, 31.2, 42.3, 43.1, 118.6, 141.6,
3332 (NH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=1.22 150.2, 153.6, 155.6, 174.2ppm; HR-MS-ESI: m/z=351.0739
(t, J=6.0Hz, 3H, CH3), 1.24–1.26 (m, 6H, CH2), 1.49 (t, ([M+K]+, Calcd), 351.0752 (Found).
J=7.2Hz, 2H, CH2), 1.80 (t, J=7.2Hz, 2H, CH2), 2.24 (t,
4-(2-Chloro-6-phenylamino-9H-purin-9-yl)-N-hydroxybu-
J=7.2Hz, 2H, CH2), 4.03 (t, J=6.6Hz, 2H, CH2), 4.13 (t, tanamide (7e′, C15H15ClN6O2)
J=6.6Hz, 2H, CH2), 6.75 (d, J=9.0Hz, 2H, ArH), 7.52 (d,
Isolated yield: 43%; white powder; mp: 208–209°C; IR
J=8.4Hz, 2H, ArH), 8.27 (s, 1H, CH), 9.29 (s, 1H, NH), 10.00 (KBr): ν=1643 (CO-acetyl), 2939 (CH-aliph), 3020 (CH-aryl),
1
(s, 1H, OH) ppm.
3290 (NH), 3422 (OH) cm−1; H-NMR (600MHz, DMSO-d6)
General Synthetic Method of Compounds 7a′–7p′ At δ=2.04–2.07 (m, 2H, CH2), 2.26 (t, J=6.0Hz, 2H, CH2), 4.20
room temperature, to a solution of hydroxylamine hydrochlo- (t, J=6.0Hz, 2H, CH2), 7.10 (t, J=9.0Hz, 1H, ArH), 7.36 (t,
ride (2.0mmol) in anhydrous methanol (20mL), 4mmol new J=9.0Hz, 2H, ArH), 7.84 (d, J=6.0Hz, 2H, ArH), 8.33 (s, 1H,
sodium methoxide was dropped into methanol. After stirring CH), 10.29 (s, 1H, NH), 12.16 (s, 1H, OH) ppm; 13C-NMR
for 30min at room temperature, compounds 6 (1.0mmol) were (151MHz, DMSO-d6) δ=24.9, 28.9, 43.7, 119.3, 121.2, 121.7,
added, and the mixture was stirred for 12h at 60°C. Upon 123.9, 128.9, 129.0, 139.4, 151.3, 152.8, 175.0ppm; HR-MS-
completion, most of the methanol was evaporated, the residue ESI: m/z=347.1023 ([M+H]+, Calcd), 347.1018 (Found).
was adjusted to pH 5–6 with HCl (1mol/L). The solution was
4-(2-Chloro-6-(4-methylphenyl)amino-9H-purin-9-yl)-N-
concentrated under reduced pressure and the crude product hydroxylbutanamide (7f′, C16H17ClN6O2)
was purified by chromatography on a silica gel column (meth-
anol/ EtOAc, 1:20) to give compounds 7a′–7p′.
Isolated yield: 41%; white powder; mp: 224–226°C; IR
(KBr): ν=1618 (CO-acetyl), 2889 (CH-aliph), 3254 (NH),
4-(2,6-Dichloro-9H-purin-9-yl)-N-hydroxybutanamide (7a′, 3365 (OH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=2.04
C9H9Cl2N5O2)
(t, J=6.0Hz, 2H, CH2), 2.18 (s, 2H, CH2), 2.28 (d, J=12Hz,
Isolated yield: 67%; white powder; mp: 90–92°C; IR 3H, CH3), 4.19 (t, J=6.0Hz, 2H, CH2), 7.17 (d, J=6.0Hz, 2H,
(KBr): ν=1570 (CO-acetyl), 2959 (CH-aliph), 3094 (CH-aryl), ArH), 7.69 (d, J=12Hz, 2H, ArH), 8.31 (s, 1H, CH), 10.20 (s,
3453 (OH) cm−1; 1H-NMR (600MHz, DMSO-d6) δ=2.05 1H, NH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=25.2, 30.8,
(t, J=9.0Hz, 2H, CH2), 2.25 (t, J=9.0Hz, 2H, CH2), 4.23 49.1, 55.7, 114.3, 119.1, 123.7, 132.2, 142.5, 151.2, 153.1, 156.2,
(t, J=9.0Hz, 2H, CH2), 8.41 (s, 1H, CH), 12.12 (s, 1H, OH) 173.0ppm; HR-MS-ESI: m/z=361.1180 ([M+H]+, Calcd),
ppm; 13C-NMR (151MHz, DMSO-d6) δ=25.2, 31.1, 60.4, 361.1167 (Found).
116.3, 142.0, 151.1, 153.5, 156.3, 172.5ppm; HR-MS-ESI:

Vol. 66, No. 4 (2018)
Chem. Pharm. Bull.
449
4-(2-Chloro-6-(4-methoxyphenyl)amino-9H-purin-9-yl)-N-
hydroxylbutanamide (7g′, C16H17ClN6O3)
8-(2-Chloro-6-phenylamino-9H-purin-9-yl)-N-hydroxyocta-
namide (7l′, C19H23ClN6O2)
Isolated yield: 52%; light yellow powder; mp: 90–91°C;
Isolated yield: 43%; white powder; mp: 158–160°C; IR
IR (KBr): ν=1622 (CO-acetyl), 3050 (CH-aryl), 2954 (CH- (KBr): ν=1670 (CO-acetyl), 2989 (CH-aliph), 3080 (CH-aryl),
1
aliph), 3258 (NH), 3566 (OH) cm−1; 1H-NMR (600MHz, 3223 (NH), 3395 (OH) cm−1; H-NMR (600MHz, DMSO-
DMSO-d6) δ=2.09 (t, J=9.0Hz, 2H, CH2), 2.36 (t, J=6.0Hz, d6) δ=1.24–1.31 (m, 6H, CH2), 1.47 (t, J=6.0Hz, 2H, CH2),
2H, CH2), 3.56 (s, 3H, CH3), 4.21 (t, J=6.0Hz, 2H, CH2), 1.81 (t, J=6.0Hz, 2H, CH2), 2.18 (t, J=6.0Hz, 2H, CH2), 4.16
7.10 (t, J=9.0Hz, 1H, NH), 7.35–7.36 (m, 2H, ArH), 7.84 (d, (t, J=9.0Hz, 2H, CH2), 7.10 (t, J=9.0Hz, 1H, ArH), 7.36 (t,
J=6.0Hz, 2H), 8.32 (s, 1H, CH), 10.26 (s, 1H, NH) ppm; J=9.0Hz, 2H, ArH), 7.84 (d, J=6.0Hz, 2H, ArH), 8.35 (s, 1H,
13C-NMR (151MHz, DMSO-d6) δ=25.2, 30.8, 51.9, 55.7, 114.3, CH), 9.86 (s, 1H, NH), 10.28 (s, 1H, NH), 11.98 (s, 1H, OH)
119.1, 123.6, 132.2, 142.5, 151.2, 153.0, 156.2, 173.0ppm; HR- ppm; 13C-NMR (151MHz, DMSO-d6) δ=24.9, 26.3, 28.6, 28.9,
MS-ESI: m/z=376.1051 ([M]+, Calcd), 376.1053 (Found).
29.6, 34.1, 43.7, 119.3, 121.2, 121.7, 123.9, 128.9, 129.0, 139.4,
4-(2-Chloro-6-(4-chlorophenyl)amino-9H-purin-9-yl)-N- 151.3, 152.9, 174.9ppm; HR-MS-ESI: m/z=402.1571 ([M]+,
hydroxybutanamide (7h′, C15H14Cl2N6O2)
Calcd), 402.1575 (Found).
Isolated yield: 38%; white powder; mp: >300°C; IR (KBr):
8-(2-Chloro-6-(4-methylphenyl)amino-9H-purin-9-yl)-N-
ν=1635 (CO-acetyl), 2851 (CH-aliph), 2930 (CH-aryl), 3173 hydroxyoctanamide (7m′, C20H25ClN6O2)
(NH), 3433 (OH) cm−1; 1H-NMR (600MHz, DMSO-d6)
Isolated yield: 46%; white powder; mp: 140–142°C; IR
δ=2.06 (t, J=9.0Hz, 2H, CH2), 2.26 (t, J=6.0Hz, 2H, CH2), (KBr): ν=1658 (CO-acetyl), 2932 (CH-aliph), 3024 (CH-aryl),
1
4.20 (t, J=9.0Hz, 2H, CH2), 7.42 (d, J=6.0Hz, 2H, ArH), 3256 (NH), 3332 (OH) cm−1; H-NMR (600MHz, DMSO-
7.89 (d, J=6.0Hz, 2H, ArH), 8.35 (s, 1H, CH), 10.43 (s, 1H, d6) δ=1.24–1.30 (m, 6H, CH2), 1.47 (t, J=6.0Hz, 2H, CH2),
NH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=25.5, 31.8, 43.4, 1.79 (t, J=9.0Hz, 2H, CH2), 2.17–2.18 (m, 2H, CH2), 2.28 (d,
119.4, 123.1, 127.6, 128.9, 138.4, 143.1, 151.5, 152.6, 165.6, J=12.0Hz, 3H, CH3), 4.15 (t, J=9.0Hz, 2H, CH2), 7.17 (d,
174.8ppm; HR-MS-ESI: m/z=379.0477 ([M−H]−, Calcd), J=6.0Hz, 2H, ArH), 7.69 (d, J=12.0Hz, 2H, ArH), 8.32 (s,
379.0475 (Found).
4-(2-Chloro-6-(4-hydroxyphenyl)amino-9H-purin-9-yl)-N- (151MHz, DMSO-d6) δ=24.9, 26.3, 28.6, 28.9, 29.9, 34.1,
1H, CH), 10.18 (s, 1H, NH), 11.96 (s, 1H, OH) ppm; 13C-NMR
hydroxybutanamide (7i′, C15H15ClN6O3)
43.7, 55.7, 114.3, 119.0, 123.7, 132.2, 142.5, 151.1, 153.1, 156.2,
Isolated yield: 41%; light yellow powder; mp: 230–232°C; 174.9ppm; HR-MS-ESI: m/z=416.1728 ([M]+, Calcd), 416.1725
IR (KBr): ν=1673 (CO-acetyl), 2741 (CH-aliph), 2929 (CH- (Found).
aryl), 3119 (NH), 3256 (OH) cm−1; 1H-NMR (600MHz,
8-(2-Chloro-6-(4-methoxyphenyl)amino-9H-purin-9-yl)-N-
DMSO-d6) δ=1.99–2.05 (m, 2H, CH2), 2.26 (t, J=9.0Hz, hydroxyoctanamide (7n′, C20H25ClN6O3)
2H, CH2), 4.17 (t, J=6.0Hz, 2H, CH2), 6.75 (t, J=6.0Hz, 2H,
Isolated yield: 40%; white powder; mp: 210–212°C; IR
ArH), 7.53 (d, J=6.0Hz, 2H, ArH), 8.26 (s, 1H, CH), 9.30 (s, (KBr): ν=1643 (CO-acetyl), 2895 (CH-aliph), 3011 (CH-aryl),
1
1H, NH), 10.01 (s, 1H, NH), 12.17 (s, 1H, OH) ppm; 13C-NMR 3300 (NH), 3516 (OH) cm−1; H-NMR (600MHz, DMSO-d6)
(151MHz, DMSO-d6) δ=25.3, 31.3, 60.3, 115.5, 119.0, 124.1, δ=1.24–1.30 (m, 6H, CH2), 1.47 (t, J=6.0Hz, 2H, CH2), 1.79
126.5, 130.6, 142.3, 151.1, 153.0, 154.5, 170.9, 174.2ppm; HR- (t, J=6.0Hz, 2H, CH2), 2.18 (t, J=6.0Hz, 2H, CH2), 3.76 (s,
MS-ESI: m/z=362.0894 ([M]+, Calcd), 362.0891 (Found).
3H, CH3), 4.14 (t, J=6.0Hz, 2H, CH2), 6.95 (d, J=6.0Hz, 2H,
8-(2,6-Dichloro-9H-purin-9-yl)-N-hydroxyoctanamide (7j′, ArH), 7.68 (d, J=6.0Hz, 2H, ArH), 8.30 (s, 1H, CH), 10.14 (s,
C13H17Cl2N5O2)
1H, NH), 11.98 (s, 1H, OH) ppm; 13C-NMR (151MHz, DMSO-
Isolated yield: 56%; mp: 76–78°C; IR (KBr): ν=1604 d6) δ=24.9, 26.3, 28.6, 28.9, 29.7, 34.1, 43.7, 55.8, 114.3, 119.0,
(CO-acetyl), 2951 (CH-aliph), 3113 (NH), 3452 (OH) cm−1; 123.7, 132.2, 142.5, 151.1, 152.9, 153.1, 156.2, 175.0ppm; HR-
1H-NMR (600MHz, DMSO-d6) δ=1.22–1.28 (m, 6H, CH2), MS-ESI: m/z=432.1677 ([M]+, Calcd), 432.1687 (Found).
1.46 (t, J=6.0Hz, 2H, CH2), 1.77 (t, J=6.0Hz, 2H, CH2), 2.16
8-(2-Chloro-6-(4-chlorophenyl)amino-9H-purin-9-yl)-N-
(t, J=6.0Hz, 2H, CH2), 4.12 (t, J=6.0Hz, 2H, CH2), 8.08 hydroxyoctanamide (7o′, C19H22Cl2N6O2)
(s, 1H, CH), 12.23 (s, 1H, OH) ppm; 13C-NMR (151MHz,
Isolated yield: 32%; white powder; mp: 190–192°C; IR
DMSO-d6) δ=15.0, 25.2, 30.8, 35.4, 42.9, 51.8, 118.7, 141.6, (KBr): ν=1636 (CO-acetyl), 2916 (CH-aliph), 3061 (CH-aryl),
1
150.3, 153.7, 155.4, 173.0ppm; HR-MS-ESI: m/z=346.0838 3138 (NH), 3342 (OH) cm−1; H-NMR (600MHz, DMSO-
([M+H]+, Calcd), 346.0839 (Found).
d6): δ=1.24–1.30 (m, 6H, CH2), 1.47 (t, J=6.0Hz, 2H, CH2),
8-(2-Chloro-6-propylamino-9H-purin-9-yl)-N-hydroxyoc- 1.81 (t, J=6.0Hz, 2H, CH2), 2.18 (t, J=9.0Hz, 2H, CH2), 4.16
tanamide (7k′, C16H25ClN6O2) (t, J=6.0Hz, 2H, CH2), 7.42 (d, J=12Hz, 2H, ArH), 7.88 (d,
Isolated yield: 41%; white powder; mp: 110–111°C; IR J=12Hz, 2H, ArH), 8.37 (s, 1H, CH), 10.42 (s, 2H, NH), 11.97
(KBr): ν=1635 (CO-acetyl), 2895 (CH-aliph), 2987 (CH-aryl), (s, 2H, OH) ppm; 13C-NMR (151MHz, DMSO-d6) δ=24.9,
1
3254 (NH), 3360 (OH) cm−1; H-NMR (600MHz, DMSO-d6) 26.3, 28.6, 28.9, 29.6, 34.2, 49.1, 119.4, 122.6, 123.1, 127.6,
δ=0.89 (t, J=6.0Hz, 3H, CH3), 1.22–1.29 (m, 6H, CH2), 1.45 128.9, 138.4, 143.1, 151.5, 152.7, 174.9ppm; HR-MS-ESI:
(t, J=9.0Hz, 2H, CH2), 1.59 (q, J=6.0Hz, 2H, CH2), 1.76 (t, m/z=436.1181 ([M]+, Calcd), IR (KBr): ν=436.1195 (Found).
J=9.0Hz, 2H, CH2), 2.17 (t, J=6.0Hz, 2H, CH2), 3.37 (q,
8-(2-Chloro-6-(4-hydroxyphenyl)amino-9H-purin-9-yl)-N-
J=6.0Hz, 2H, CH2), 3.81 (s, 1H, NH), 4.08 (t, J=6.0Hz, 2H, hydroxyoctanamide (7p′, C19H23ClN6O3)
CH2), 8.15 (s, 1H, CH), 8.26 (s, 1H, NH), 11.97 (s, 1H, OH)
Isolated yield: 35%; white powder; mp: 230–232°C. IR
ppm; 13C-NMR (151MHz, DMSO-d6) δ=11.8, 22.6, 24.9, (KBr): ν=1636 (CO-acetyl), 2972 (CH-aliph), 3060 (CH-aryl),
26.3, 28.9, 34.1, 42.3, 43.5, 118.6, 141.6, 150.2, 153.6, 155.6, 3240 (OH) cm−1; 1H-NMR (600MHz, DMSO-d6): δ=1.23–1.30
174.9ppm; HR-MS-ESI: m/z=369.1806 ([M+H]+, Calcd), (m, 6H, CH2), 1.47 (t, J=6.0Hz, 2H, CH2), 1.79 (t, J=9.0Hz,
369.1807 (Found).
2H, CH2), 2.18 (t, J=6.0Hz, 2H, CH2), 4.13 (t, J=9.0Hz, 2H,

450
Chem. Pharm. Bull.
Vol. 66, No. 4 (2018)
CH2), 6.75 (d, J=6.0Hz, 2H, ArH), 7.51 (d, J=12.0Hz, 2H, the membranes were saturated with enhanced chemilumines-
ArH), 8.27 (s, 1H, CH), 9.29 (s, 1H, NH), 10.00 (s, 1H, NH), cence mixture for 1min, and chemiluminescence was viewed
11.97 (s, 1H, OH) ppm; 13C-NMR (151MHz, DMSO-d6): by autography using pre-flashed X-ray film (FUJIFILM,
δ=24.9, 26.3, 28.6, 28.9, 29.7, 34.2, 43.7, 115.5, 119.0, 124.0, Tokyo, Japan) for 300s.
130.6, 142.3, 151.0, 153.0, 153.2, 154.4, 175.0ppm; HR-MS-
Docking Studies Docking studies were performed using a
ESI: m/z=441.1418 ([M+Na]+, Calcd), 441.1405 (Found).
free Autodock 4.0.39) The three-dimensional structures of the
In Vitro HDAC Assay The HDAC Colorimetric Assay/ proposed compounds were constructed and energy minimiza-
Drug Discovery Kit was bought from Enzo Biochem Inc. The tions were performed with the Chem-Draw/Chem3D. Docking
reagents were prepared for assay following the instructions. studies were performed as described in our previous papers.35)
On the 96-well plate, HDACs (5µL/well) were incubated at The complexes pictures were rendered employing the UCSF
37°C with 10µL of various concentrations of inhibitors and Chimera software.40)
25µL of substrate. After reacting for 30min, Color de Lys
Developer (50µL/well) was added. The ultraviolet absorption
Acknowledgments This work was supported by the
of the wells was measured on a microtiter-plate reader (BIO- National Nature Science Fundation of China (Grant No.
RAD: model 680) at 405nm after 15min. The inhibition ratios 81360469) and the program of Science and Technology Project
were calculated from the optical density (OD) values. Finally, of Jiangxi Province (20122BBG70094-2). The authors also
the IC50 values were determined using a regression analysis of thank Center of Analysis and Testing of Nanchang University
the concentration/inhibition data.
for assistance with the MS and NMR testing of compounds.
MTT Assay Antitumor activity in vitro was determined
by the improved MTT assay.32) The HCT116, K562 cell lines
Conflict of Interest The authors declare no conflict of
were cultured in RPMI1640 medium containing 10% heat- interest.
inactivated fetal bovine serum (FBS) at 37°C in 5% CO2 hu-
midified incubator. The logarithmic growth phase cells were
Supplementary Materials The online version of this ar-
collected for experiments. Tumor cells (2.0×105 cells /mL) ticle contains supplementary materials.
were inoculated in 96-well culture plates (90µL/well). Then
10 µL of culture medium containing synthetic compound of
various concentrations was added to the wells, then the cells
were incubated for 48h at 37°C in 5% CO2 atmosphere. Twen-
ty microliters of MTT was added at a final concentration of
5mg/mL and after 4h incubation, 100µL Triple solution (10%
sodium dodecyl sulfate (SDS), 5% isobutanol, 0.01mol/L HCl)
were added. The suspension was placed in the dark incubator
at 37°C overnight and the optical density was measured at
570nm, then the IC50 values were calculated.
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