Synthesis and evaluation of novel carbocyclic oxetanocin a (COA-Cl) derivatives as potential tube formation agents

Article abstract of DOI:10.1248/cpb.c15-00386

Six novel carbocyclic oxetanocin A analogs (2-chloro-C.OXT-A; COA-Cl) with various hydroxymethylated or spiro-conjugated cyclobutane rings at the N⁹-position of the 2-chloropurine moiety were synthesized and evaluated using human umbilical vein

Full text of DOI:10.1248/cpb.c15-00386

Vol. 63, No. 9
Chem. Pharm. Bull. 63, 701–709 (2015)
701
Regular Article
Synthesis and Evaluation of Novel Carbocyclic Oxetanocin A (COA-Cl)
Derivatives as Potential Tube Formation Agents
,a
Norikazu Sakakibara,* Junsuke Igarashi,b Maki Takata,c Yosuke Demizu,d Takashi Misawa,d
Masaaki Kurihara,d Ryoji Konishi,c Yoshihisa Kato,a Tokumi Maruyama,a and Ikuko Tsukamotoc
a Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University; 1314–1 Shido, Sanuki,
b
Kagawa 769–2193, Japan: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University;
c
1750–1 Ikenobe, Miki, Kita, Kagawa 761–0793, Japan: Department of Pharmaco-Bio-Informatics, Faculty of
Medicine, Kagawa University; 1750–1 Ikenobe, Miki, Kita, Kagawa 761–0793, Japan: and d Division of Organic
Chemistry, National Institute of Health Sciences; 1–1–18 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan.
Received May 6, 2015; accepted June 29, 2015
Six novel carbocyclic oxetanocin A analogs (2-chloro-C.OXT-A; COA-Cl) with various hydroxy-
methylated or spiro-conjugated cyclobutane rings at the N9-position of the 2-chloropurine moiety were
synthesized and evaluated using human umbilical vein endothelial cells. All prepared compounds
(2a–f) showed good to moderate activity with angiogenic potency. Among these compounds, 100µM cis–
trans-2′,3′-bis(hydroxymethyl)cyclobutyl derivative (2b), trans-3′-hydroxymethylcyclobutyl analog (2d), and
3′,3′-bis(hydroxymethyl)cyclobutyl derivative (2e) had greater angiogenic activity, with relative tube areas of
3.43±0.44, 3.32±0.53, and 3.59±0.83 (mean±standard deviation (S.D.)), respectively, which was comparable
to COA-Cl (3.91±0.78). These data may be important for further development of this class of compounds as
potential tube formation agents.
Key words 2-chloro-carbocyclic oxetanocin A; 2-chloro-C.OXT-A; nucleoside derivative; angiogenic activity
Angiogenesis, the process in which new capillary vessels Buerger’s disease, or peripheral arterial obliterative disease,
are built from preexisting ones, plays an important role in a and could be developed as an adhesive skin patch, lotion, or
variety of situations, including wound healing, tumor growth, oral agent. In addition to its angiogenic effect, we found that
and metastasis. Angiogenesis promoters are essential for de- COA-Cl protects against oxygen-glucose deprivation in pri-
tecting various symptoms arising due to a lack of blood flow, mary cortical neurons in a manner sensitive to suramin.7) Be-
such as Buerger‘s disease or chronic arteriosclerosis obliterans cause of its utility, COA-Cl is commercially available (Chemi-
in diabetic patients. However, the clinical applications of these cal Name: 2-Cl-C.OXT-A, WAKENYAKU Co., Ltd., Japan,
promoters as medical agents remain minimal compared to Order Number: 032-21541, Quantity: 1mg, Price: 15000 yen).
angiogenesis inhibitors, which are utilized as antitumor thera-
Our mechanistic studies revealed that the angiogenic effects
peutics. The lack of angiogenesis promoters used as therapeu- of COA-Cl involves endothelial S1P1 receptor. S1P1 recep-
tic agents may be due to limited knowledge of these promoter, tor is activated by a serum-borne lipid mediator sphingosine
with the exception of growth factors derived from living sys- 1-phosphate (S1P) as a natural ligand, coupled with G-protein
tems. For example, vascular endothelial growth factor (VEGF) α i/o subunit, and is indispensable during normal vascular
and fibroblast growth factor (FGF) are macromolecular glyco- development.8) Stimulation of S1P1 by COA-Cl activates intra-
proteins that are chemically and biologically unstable. Hence, cellular signaling pathways, ultimately inducing tube forma-
they are used only in treatments administered by injection or tion in human umbilical vein endothelial cells (HUVECs).9)
gene transfection approaches.1–3)
In agreement with our previous reports, COA-Cl promotes the
We synthesized an angiogenesis promoter 2-chloro- phosphorylation and activation of the mitogen activated pro-
carbocyclic oxetanocin A (COA-Cl), (1a, racemate) (Fig. 1), tein kinases (MAPK), extracellular signaling-related kinases
which is the first low-molecular weight active compound in (ERK1/2), which induce angiogenesis and tube formation, a
this class.4–6) The size of compound 1a (molecular weight hallmark of HUVEC angiogenesis.4) The ability of COA-Cl to
(MW)=284) is suitable for pharmaceutical use in percutane- modulate ERK1/2 phosphorylation and tube formation is simi-
ous and transmucosal absorption. Therefore, it is expected to lar to S1P (Fig. 2), the naturally occurring ligand for the S1P1
have practical applications as a therapeutic for wound healing, receptor.10–12) Thus, these results indicate that COA-Cl is a
Fig. 1. Structures of COA-Cl (1a), COA-Oe (1b), C.OXT-A (1c), Cladribine (1d)
*To whom correspondence should be addressed. e-mail: bara@kph.bunri-u.ac.jp
© 2015 The Pharmaceutical Society of Japan

702
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
Fig. 2. Structures of S1P, FTY-720, FTY-720-P, KRP-203, and ACT-128800
Fig. 3. Structures of COA-Cl (1) and COA-Cl Analogs (2a–f)
novel and potent angiogenic agent that may behave as a partial COA-Cl 1a was essential for its angiogenic activity. Neverthe-
agonist of the S1P1 receptor. less, the role of the two hydroxymethyl groups bound to the
Several S1P derivatives have been developed as S1P1 ago- cyclobutane ring at the 2′ and 3′ position remain unclear.
nists, including FTY-720,13,14) FTY-720-P,13,14) KRP-203,15) and
In the present study, we sought to further elucidate the an-
ACT-12880016) (Fig. 2). These compounds can block the egress giogenic function of hydroxymethyl group(s) on COA-Cl 1a.
of T cells from the thymus and lymphoid organs,17,18) and may More specifically, six novel COA-Cl analogs racemates 2a,
be promising oral therapeutics for autoimmune disorders. b, and achiral compounds 2c–f (Fig. 3) with a variety of hy-
Nevertheless, outside of COA-Cl 1a, no other compound has droxymethylated or spiro-conjugated cyclobutane rings at the
been shown to exert neovascularization effects via the S1P1 N9-position in the 2-chloropurine moiety were synthesized and
receptor. Thus, COA-Cl has the potential for clinical applica- evaluated in HUVECs.
tions as an angiogenic medicine acting via the S1P1 receptor.
Understanding of the unique mechanism of COA-Cl 1a has
Results and Discussion
triggered intensive effort to discover COA-Cl analogs with
Chemistry The COA-Cl analogs 2a–d shown in Chart
enhanced angiogenic activity and reduced cytotoxicity.4,6) 1 were prepared using known cyclobutyl alcohols (3a,19) b,20)
However, our previous work has resulted in analogs with no c,21,22) d,21,23)) as starting materials in two steps reactions,
or less potency to COA-Cl 1a. For example, the COA-Cl de- according to a modified method from a previous report.24)
rivative, COA-OMe (1b), in which the 2-chloro group at the Secondary alcohols 3a–d were treated with 2,6-dichloropu-
2-position in the purine system is substituted with a methoxy rine and underwent the Mitsunobu reaction25) in the presence
group (Fig. 1), displayed half the vascularization activity of of PPh3 and DIAD to yield 9-cyclobutyl-2,6-dichloropurine
COA-Cl 1a.6) Furthermore, substitution of an H in place of the congeners, 4a–d, in 37–84% yields, which were treated with
Cl (1c: C.OXT-A) or replacement of a cyclobutane moiety with methanolic ammonia to give the desired products 2a–d in
the pentose ring (1d: cladribine, used to treat hairy cell leu- 68–87% yields.
kemia and multiple sclerosis) showed no angiogenic potency.4)
The relative configurations of 4b–d were determined on
Collectively, these studies indicated that the structure of the the basis of nuclear Overhauser effect spectroscopy (NOESY)
2-chloropurine skeleton and the cyclobutane ring moiety in correlations (Fig. 4) and comparison with previous litera-

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
703
Reagents and conditions: i, 2,6-Dichloropurine, PPh3, DIAD, THF, 50°C; ii, NH3, MeOH, 100°C.
Chart 1. Synthesis of Compounds 2a–d
Reagents and conditions: i, BzCl, Pyridine, rt; ii, Pd/C, H2, rt; iii, 2,6-Dichloropurine, PPh3, DIAD, THF, 50°C; iv, NH3, MeOH, 100°C; v, TsCl, Pyridine, rt; vi, NaH,
Dimethylmalonate, toluene, reflux; vii, LiAlH4, THF, −20°C.
Chart 2. Synthesis of Compounds 2e and f
Fig. 4. Key NOESY (H↔H) Correlations and Relative Configuration of Compounds 4b–d
ture.20–23) For compound 4b, the NOESY correlations between H-3′ indicated the 1′,3′-trans derivative (4d).
H-1′ and H-4′b, between H-4′a and H-3′, and between H-2′
The synthesis of COA-Cl derivatives 2e and f is described
and H-3′, and the absence of NOESY correlations between in Chart 2. Starting from 3,3-bis(hydroxymethyl)-1-ben-
H-1′ and H-2′, suggested the 1′,2′-trans-2′,3′-cis congener (4b). zyloxycyclobutane 5,26) we prepared dibenzoyl ester 6 in a
Similarly, for compounds 4c and d, the NOESY correlations 90% yield, followed by debenzylation with palladium carbon
between H-1′ and H-3′ indicated the 1′,3′-cis analog (4c), (Pd–C)/H2 to afford alcohol 7 in 91% yield. Compound 7
whereas the absence of NOESY correlations between H-1′ and was then condensed with 2,6-dichloropurine using the Mitsu-

704
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
Fig. 5. Pictures of Stimulated Tube Formation by 1a and 2a–f
Additives are shown under the pictures.
Fig. 6. Tube Formation Assay Using Compounds 1a and 2a–f
The tube formation assay was performed using an angiogenesis kit, and the estimation was performed 10d after incubation with the designated compounds. The area of
the formed tube was represented as a relative value versus control wells with no additives. The effect of the solvent (10% saline) and positive control (VEGF; 10ng/mL) are
shown together. Results were expressed as mean±S.E. of 2–6 individual experiments.
nobu reaction25) to afford 8 in a 75% yield, which was treated which in turn was alkylated by diisopropylmalonate to form
with methanolic ammonia to give the desired product 2e in diisopropyl dicarboxylated product 9 in an 87% yield, pos-
a 50% yield. As for the preparation of spiro-counterpart 2f, sessing the spiro[3.3]heptane core.27) The resulting product 9
compound 5 was transformed into its (bis)tosyl derivative, was reduced with LiAlH4 to afford diol 10 in a 34% yield,

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
705
Fig. 7. Effect of Compounds 2a–f on the Proliferation
Forty-eight hours after the addition of additives, the cell viability was measured with a Cell Counting Kit-8. Cell viabilities were represented as relative values to that
of the control well with no additive. The effect of the solvent, 10% saline as well as a positive control, VEGF (10ng/mL) were shown together. Results were expressed as
mean±S.E. of more than four individual experiments. *p<0.01 and # p<0.05 vs. solvent (by Student’s t-test).
which was subsequently acylated with benzoyl chloride to plex cellular processes, including proliferation of endothelial
yield dibenzoyl ester 11 in an 87% yield. Compound 11 was cells,4,28,29) which showed that compounds 2a–f promoted the
hydrogenated with Pd–C to produce secondary alcohol 12 in proliferation of HUVECs at a higher concentration (e.g., 2a:
a 72% yield, followed by the Mitsunobu reaction25) to provide 200µM: 1.12±0.07, n=5, p=0.031), whereas 2f at a concen-
the spiro-condensation product 13 in an 86% yield, which was tration of 10µM only inhibited the proliferation of HUVECs
treated with methanolic ammonia to give the objective spiro- (0.72±0.19, n=5, p=0.005).
diol 2f in a 70% yield.
Biological Activity The effects of the synthesized nu-
cleoside analogs (2a–f) on angiogenesis were examined. We
Conclusion
In the present study, a series of newly synthesized COA-Cl
evaluated the analogs using a well-established tube formation analogs exhibited good to moderate angiogenic activity. In
assay.4,28,29) All experiments were performed 2–6 times. Ten particular, 100µM of the COA-Cl analogs, 2b, d, and e induced
days following the incubation of the fibroblasts and additives, enhanced angiogenesis, with relative tube areas of 3.43±0.44,
HUVECs were stained using the Tubule Staining Kit for 3.32±0.53, and 3.59±0.83, respectively. This was comparable
CD31. Typical pictures are shown in Fig. 5. VEGF was used with the tube area induced by COA-Cl (3.91±0.78). Overall,
as a positive control. The area of the formed tube was rep- our data may serve as the basis for further modification to
resented as a relative value to tubes formed in wells without identify more potent candidates to induce tube formation.
additives (saline). Figure 6 shows the dose-dependent tube for-
mation responses, as well as the solvent control (10% saline)
and positive control (VEGF, 10ng/mL).
Experimental
Chemistry 1H-NMR and 13C-NMR spectra were taken
As shown in Figs. 5 and 6, angiogenic potencies were with a Ultrashield™ 400 Plus FT NMR System (BRUKER).
observed in every prepared compound, including the cis– Chemical shifts and coupling constants (J) were given in δ
trans-2′,3′-bis(hydroxymethyl)cyclobutyl derivative, 2a, cis– and Hz, respectively. Melting points were determined on a
trans-2′,3′-bis(hydroxymethyl)cyclobutyl derivative, 2b, cis- Yanaco MP-500D. High-resolution mass spectrometry was
3′-hydroxymethylcyclobutyl derivative, 2c, trans-3′-hydroxy- performed on a APEX IV mass spectrometer (BRUKER) with
methylcyclobutyl derivative, 2d, 3′,3′-bis(hydroxymethyl)- electrospray ionization mass spectroscopy (ESI-MS).
cyclobutyl derivative 2e, and 2′,2′-bis(benzoyloxymethyl)-
General Procedure for the Synthesis of 4a–d A solution
spiro[3′.3′]hept-6′-yl derivative 2f (Fig. 3). Among these, of compound 3a–d (0.38mmol), triphenylphosphine (104.9mg,
at a concentration of 100µM, 2b, d, and e exerted greater 0.40mmol), 2,6-dichloropurine (75.4mg, 0.40mmol) and
angiogenic activity (3.43±0.44, 3.32±0.53, and 3.59±0.83 DIAD (diisopropyl azodicarbosylate, 78.8µL, 0.40mmol) in
[mean±standard deviation (S.D.)], respectively) which was tetrahydrofuran (THF) (3.0mL) was stirred at 50°C. After
comparable to COA-Cl 1a (3.91±0.78). Compound 2d was 8–15h stirring, the residual solution was purified by silica gel
most angiogenic when using 10µM (2.74±0.28), which was column chromatography (60% AcOEt in hexane) to give 4a–d.
superior to the potency of COA-Cl 1a (2.44±0.89 at 10µM) in
2,6-Dichloro-9-[cis–trans-2′,3′-bis(benzoyloxymethyl)-
our previous report4) and 10ng/mL VEGF (2.13±0.61). Sur- cyclobutyl]purine (4a)
1
prisingly, the angiogenic activity of spiro analog 2f at 10µM
Yield 37%; oil; H-NMR (400MHz, CDCl3) δ: 8.34 (1H,
was 2.42±0.46, which was nearly identical to that of 1a at s, H-8), 8.06–8.12 (2H, m, Bz), 7.45–7.64 (6H, m, Bz),
same concentration. The potency of 2d–f reached a maximum 7.31–7.38 (2H, m, Bz), 5.52 (1H, m, H-1′), 4.53–4.66 (2H, m,
around 10–100µM, and decreased at 100–200µM, indicating 3′-CH2OBz), 4.30–4.41 (2H, m, 2′-CH2OBz), 3.42 (1H, m,
that the cellular proliferation was eventually inhibited at a H-2′), 3.20 (1H, m, H-4′a), 2.96 (1H, m, H-3′), 2.76 (1H, m,
high analog concentration.
H-4′b); 13C-NMR (100MHz, CDCl3) δ: 166.4, 165.5, 153.4,
Moreover, as shown in Fig. 7, we performed a proliferation 153.0, 151.8, 144.2, 144.2, 133.4, 133.4, 131.0, 129.6, 129.0,
assay because angiogenesis is intimately associated with com- 128.7, 128.6, 128.5, 65.9, 62.4, 48.8, 41.4, 32.3, 26.9; high reso-

706
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
lution (HR)-MS (ESI) Calcd for C25H20Cl2N4NaO4 [M+Na]+: 209.1–210.5°C.
533.07538. Found 533.0713. 2-Amino-6-chloro-9-(cis-3′-hydroxymethylcyclobutyl)-
2,6-Dichloro-9-[trans–cis-2′,3′-bis(benzoyloxymethyl)- purine (2c)
cyclobutyl]purine (4b)
Yield 69%; white crystals; 1H-NMR (400MHz, CD3OD)
1
Yield 38%; white crystals; H-NMR (400MHz, CDCl3) δ: δ: 8.22 (1H, s, H-8), 4.90 (1H, m, H-1′), 3.67 (2H, m,
8.28 (1H, s, H-8), 8.08 (2H, m, Bz), 7.69 (2H, m, Bz), 7.30–7.63 3′-CH2OBz), 2.66 (2H, m, H-2′a and H-4′a), 2.44 (3H, m,
(6H, m, Bz), 5.25 (1H, m, H-1′), 4.52–4.75 (4H, m, 2′-CH2OBz H-2′b, H-3′ and H-4′b); 13C-NMR (100MHz, CD3OD) δ:
and 3′-CH2OBz), 3.73 (1H, m, H-2′), 3.17 (1H, m, H-3′), 2.99 158.0, 155.0, 151.9, 141.3, 119.2, 66.1, 46.8, 33.3, 31.7; HR-MS
(1H, m, H-4′a), 2.58 (1H, m, H-4′b); 13C-NMR (100MHz, (ESI) Calcd for C10H12ClN5NaO [M+Na]+: 276.06226. Found
CDCl3) δ: 166.3, 166.2, 152.9, 152.8, 151.8, 144.4, 133.4, 133.4, 276.06236; mp: 152.3–153.4°C.
131.1, 129.6, 129.6, 129.2, 129.0, 128.6, 128.4, 64.0, 63.1, 51.5,
43.4, 30.2, 29.2; HR-MS (ESI) Calcd for C25H20Cl2N4NaO4 purine (2d)
[M+Na]+: 533.07538. Found 533.07531; mp: 57.5–58.5°C.
Yield 68%; white crystals; H-NMR (400MHz, CD3OD) δ:
2,6-Dichloro-9-(cis-3′-benzoyloxymethylcyclobutyl)purine 8.17 (1H, s, H-8), 5.00 (1H, m, H-1′), 3.64 (2H, m, 3′-CH2OBz),
2-Amino-6-chloro-9-(trans-3′-hydroxymethylcyclobutyl)-
1
(4c)
2.67 (2H, m, H-2′a and H-4′a), 2.52 (3H, m, H-2′b, H-3′ and
Yield 62%; white crystals; 1H-NMR (400MHz, CDCl3) H-4′b); 13C-NMR (100MHz, CD3OD) δ: 158.0, 155.1, 151.9,
δ: 8.20 (1H, s, H-8), 8.05 (2H, m, Bz), 7.59 (1H, m, Bz), 141.4, 119.3, 65.7, 48.6, 32.3, 32.1; HR-MS (ESI) Calcd for
7.47 (2H, m, Bz), 5.00 (1H, m, H-1′), 4.47 (2H, d, J=5.2Hz, C10H12ClN5NaO [M+Na]+: 276.06226. Found 276.06239; mp:
3′-CH2OBz), 2.85 (2H, m, H-2′a and H-4′a), 2.75 (1H, m, 256.5–257.9°C.
H-3′), 2.60 (2H, m, H-2′b and H-4′b); 13C-NMR (100MHz,
3,3-Bis(benzoyloxymethyl)-1-benzyloxycyclobutane
(6)
CDCl3) δ: 166.5, 152.9, 152.8, 151.8, 144.1, 133.3, 129.9, 129.5, Compound 5 (2.48g, 11.16mmol) was dissolved in dry pyri-
129.5, 128.6, 128.6, 66.8, 46.1, 33.1, 28.0; HR-MS (ESI) Calcd dine (24.2mL), and benzoyl chloride (2.72mL, 23.44mmol)
for C17H14Cl2N4NaO2 [M+Na]+: 399.03860. Found 399.03772; was added dropwise to the solution at 0°C, and the mixture
mp: 164.2–165.6°C.
2,6-Dichloro-9-(trans-3′-benzoyloxymethylcyclobutyl)- then cooled to 0°C, 5.0mL of water was added over 3min,
purine (4d) and the mixture was stirred at room temperature for 1h. The
was stirred for 2h at room temperature. The mixture was
Yield 84%; white crystals; 1H-NMR (400MHz, CDCl3) solvent was removed in vacuo and the residue was extracted
δ: 8.25 (1H, s, H-8), 8.10 (2H, m, Bz), 7.61 (1H, m, Bz), with AcOEt. The organic extracts were washed with water,
7.49 (2H, m, Bz), 5.30 (1H, m, H-1′), 4.54 (2H, d, J=6.4Hz, saturated potassium carbonate solution and saturated sodium
3′-CH2OBz), 3.04 (1H, m, H-3′), 2.91 (2H, m, H-2′a and chloride solution, dried with sodium sulfate, and then evapo-
H-4′a), 2.70 (2H, m, H-2′b and H-4′b); 13C-NMR (100MHz, rated. The residue was purified by silica gel column chro-
CDCl3) δ: 166.6, 153.0, 152.9, 151.9, 144.2, 133.3, 131.2, 129.9, matography (30% AcOEt in hexane) to give a colorless oil
1
129.6, 128.6, 66.7, 48.1, 31.8, 28.3; HR-MS (ESI) Calcd for 6 (4.31g, 10.04mmol, 90%). H-NMR (400MHz, CDCl3) δ:
C17H14Cl2N4NaO2 [M+Na]+: 399.03860. Found 399.03753; mp: 7.99–8.08 (4H, m, Bz), 7.52–7.58 (2H, m, Bz), 7.38–7.45 (4H,
147.7–149.5°C.
m, Bz), 7.26–7.36 (5H, m, Bn), 4.44 (4H, s, 3-CH2OBz ×2),
General Procedure for the Synthesis of 2a–d Com- 4.42 (2H, s, Bn), 4.22 (1H, m, H-1), 2.40 (2H, m, H-2a and
pounds 4a–d (0.12mmol) was dissolved in NH3 (14.0 mL)/ H-4a), 2.19 (2H, m, H-2b and H-4b); 13C-NMR (100MHz,
MeOH (3.0mL), and then sealed and stirred for 1–3d at CDCl3) δ: 166.6, 166.5, 138.0, 133.2, 133.1, 130.0, 129.8, 129.7,
100°C. The mixture was evaporated, and the residue was puri- 129.6, 128.5, 128.4, 128.4, 127.9, 127.7, 70.1, 68.7, 68.4, 67.7,
fied by silica gel column chromatography (20–25% MeOH in 35.0, 34.7; HR-MS (ESI) Calcd for C27H26NaO5+ [M+Na]+:
CH2Cl2) to afford 2a–d.
453.16725. Found 453.16578.
2-Amino-6-chloro-9-[cis–trans-2′,3′-bis(hydroxymethyl)-
3,3-Bis(benzoyloxymethyl)cyclobutan-1-ol (7) To a so-
lution of this compound 6 (4.18g, 9.71mmol) in methanol
cyclobutyl]purine (2a)
1
Yield 84%; white crystals; H-NMR (400MHz, CDCl3) δ: (53.4mL) was added 10% Pd on carbon (3.24g). The reaction
8.36 (1H, s, H-8), 7.70 (2H, brs, 6-NH2), 5.07 (1H, m, H-1′), mixture was stirred at room temperature under H2 atmosphere
4.75 (1H, t, J=4.8Hz, 3′-OH), 4.30 (1H, t, J=5.2Hz, 2′-OH), (1atm) for 18h. The suspension was filtered through a pad
3.56 (2H, m, 3′-CH2OBz), 3.31 (2H, m, 2′-CH2OBz), 2.90 of Celite. The filtrate was evaporated, and then the residue
(1H, m, H-4′a), 2.58 (1H, m, H-2′), 2.33 (1H, m, H-4′b), 2.30 was purified by silica gel column chromatography (60%
(1H, m, H-3′); 13C-NMR (100MHz, CDCl3) δ: 158.0, 155.1, AcOEt in hexane) to give a colorless oil 7 (3.01g, 8.84mmol,
152.6, 142.2, 119.1, 65.4, 61.6, 50.0, 45.4, 36.5, 27.8; HR-MS 91%). 1H-NMR (400MHz, CDCl3) δ: 8.00 8.07 (4H, m,
(ESI) Calcd for C11H14ClN5NaO2 [M+Na]+: 306.07282. Found Bz), 7.51–7.58 (2H, m, Bz), 7.38–7.45 (4H, m, Bz), 4.50 (1H,
306.07162; mp: 226.5–227.7°C.
m, H-1), 4.47 (2H, s, 3-CH2OBz), 4.42 (2H, s, 3-CH2OBz),
2-Amino-6-chloro-9-[trans–cis-2′,3′-bis(hydroxymethyl)- 2.46 (2H, m, H-2a and H-4a), 2.10 (2H, m, H-2b and H-4b);
cyclobutyl]purine (2b)
13C-NMR (100MHz, CDCl3) δ: 166.6, 166.6, 133.2, 133.1,
Yield 87%; white crystals; 1H-NMR (400MHz, CD3OD) 129.8, 129.7, 129.6, 129.6, 128.5, 128.4, 68.9, 67.6, 62.7,
δ: 8.14 (1H, s, H-8), 4.80 (1H, m, H-1′), 3.65–3.80 (4H, m, 37.6, 34.2; HR-MS (ESI) Calcd for C20H20NaO5+ [M+Na]+:
2′-CH2OBz and 3′-CH2OBz), 3.20 (1H, m, H-2′), 2.62 (1H, 363.12029. Found 363.11926.
m, H-4′a), 2.59 (1H, m, H-3′), 2.29 (1H, m, H-4′b); 13C-NMR
2,6-Dichloro-9-[3′,3′-bis(benzoyloxymethyl)cyclobu-
(100MHz, CD3OD) δ: 158.0, 155.0, 152.0, 141.7, 119.3, tyl]purine (8) solution of compound (204.2mg,
A
7
62.6, 61.6, 52.0, 47.3, 34.2, 29.7; HR-MS (ESI) Calcd for 0.60mmol), triphenylphosphine (157.4mg, 0.60mmol), 2,6-di-
C11H14ClN5NaO2 [M+Na]+: 306.07282. Found 306.07305; mp: chloropurine (94.5mg, 0.50mmol) and TMAD (N,N,N′,N′-

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
707
tetramethylazodicarboxamide, 103.3mg, 0.60mmol) in THF
6-Benzyloxy-2,2-bis(hydroxymethyl)spiro[3.3]heptane
(5.0mL) was stirred at 50°C. After 22h stirring, the residual (10) A solution of compound 9 (620.1mg, 1.66mmol) in
solution was purified by silica gel column chromatography dry THF (5.0mL) was added by drops to an ice-cooled sus-
(60% AcOEt in hexane) to give as a colorless oil 8 (192.0mg, pension of lithium aluminum hydride (103.9mg, 2.34mmol)
1
0.38mmol, 75%). H-NMR (400MHz, CDCl3) δ: 8.23 (1H, s, in dry THF (10.0mL), and the mixture was stirred for 1h at
H-8), 7.99–8.11 (4H, m, Bz), 7.56–7.64 (2H, m, Bz), 7.41–7.51 room temperature, then cooled. The solution was treated with
(4H, m, Bz), 5.27 (1H, m, H-1′), 4.64 (2H, s, 3′-CH2OBz), AcOEt (2.0mL) and water (1.0mL) to give a gel. The solid
4.63 (2H, s, 3′-CH2OBz), 2.93 (2H, m, H-2′a and H-4′a), 2.83 was filtrated and washed successively with AcOEt (100mL).
(2H, m, H-2′b and H-4′b); 13C-NMR (100MHz, CDCl3) δ: The filtrate and washing were combined, and washed with
166.5, 166.3, 152.9, 152.0, 144.1, 133.5, 133.5, 129.7, 129.6, saturated sodium chloride solution, dried with sodium sulfate,
128.6, 67.8, 66.4, 60.4, 36.8, 34.3; HR-MS (ESI) Calcd for and then evaporated. The residue was purified by silica gel
C25H20Cl2N4NaO2 [M+Na]+: 533.07538. Found 533.07375.
column chromatography (20% MeOH in AcOEt) to give as
2-Amino-6-chloro-9-[3′,3′-bis(hydroxymethyl)cyclobutyl]- an oil 10 (149.6mg, 0.56mmol, 34%). 1H-NMR (400MHz,
purine (2e) Compound 8 (192.0mg, 0.38mmol) was dis- CDCl3) δ: 7.24–7.36 (5H, m, Bn), 4.37 (2H, s, Bn), 3.94 (1H,
solved in NH3 (14.0mL)/MeOH (3.0mL), and then sealed and m, H-6), 3.64 (4H, s, 2-CH2OH ×2), 2.32 (2H, m, H-5a and
stirred for 4d at 100°C. The mixture was evaporated, and the H-7a), 2.02 (2H, m, H-5b and H-7b), 1.82 and 1.85 (4H, s, H-1
residue was purified by silica gel column chromatography and H-3); 13C-NMR (100MHz, CDCl3) δ: 138.2, 128.4, 127.9,
(25% MeOH in CH2Cl2) to give 2e as white crystals (53.7mg, 127.7, 70.1, 69.3, 68.7, 44.3, 39.6, 39.2, 38.8, 29.5; HRMS (ESI)
1
0.19mmol, 50%). H-NMR (400MHz, DMSO-d6) δ: 8.38 (1H, Calcd for C16H22NaO3 [M+Na]+: 285.14612. Found 285.14612.
s, H-8), 7.80 (1H, brs, 6-NH2), 4.94 (1H, m, H-1′), 4.88 (1H,
6-Benzyloxy-2,2-bis(benzoyloxymethyl)spiro[3.3]heptane
t, J=5.2Hz, 3′-CH2OH), 4.78 (1H, t, J=5.2Hz, 3′-CH2OH), (11) Compound 10 (151.3mg, 0.58mmol) was dissolved
3.56 (2H, d, J=5.2Hz, 3′-CH2OH), 3.49 (2H, d, J=5.2Hz, 3′- in dry pyridine (1.3mL), and benzoyl chloride (200.0µL,
CH2OH), 2.47 (2H, m, H-2′a and H-4′a), 2.39 (2H, m, H-2′b 1.73mmol) was added dropwise to the solution at 0°C, and
and H-4′b); 13C-NMR (100MHz, DMSO-d6) δ: 156.8, 152.7, the mixture was stirred for 3h at room temperature. The
150.3, 139.7, 118.0, 65.0, 64.1, 43.2, 33.0; HR-MS (ESI) Calcd mixture was then cooled to 0°C, 0.3mL of water was added
for C11H14ClN5NaO2 [M+Na]+: 306.07282. Found 306.07166; over 1min, and the mixture was stirred at room temperature
mp: 239.6°C.
for 30min. The solvent was removed in vacuo and the residue
6-Benzyloxyspiro[3.3]heptane-2,2-dicarboxylic Acid Di- was extracted with AcOEt. The organic extracts were washed
isopropyl Ester (9) Compound 5 (2.06g, 9.27mmol) was with water, saturated potassium carbonate solution and satu-
dissolved in dry pyridine (7.7mL) and cooled by an ice bath. rated sodium chloride solution, dried with sodium sulfate, and
p-Toluenesulfonyl chloride (4.42g, 23.17mmol) was added in then evaporated. The residue was purified by silica gel column
small portions to the cooled solution under stirring in a dry chromatography (30% AcOEt in hexane) to give a colorless oil
1
atmosphere. The mixture was left standing overnight. The 11 (238.2mg, 0.51mmol, 87%). H-NMR (400MHz, CDCl3) δ:
precipitate was filtered off, the filtrate was carefully poured 8.02 (4H, m, Bz), 7.55 (2H, m, Bz), 7.42 (4H, m, Bz), 7.24–7.36
into water (50mL), and the product was extracted with AcOEt. (5H, m, Bn), 4.38, 4.40 and 4.41 (6H, s, 2-CH2OBz ×2 and
The extract was washed with 1N HCl, water and saturated Bn), 3.96 (1H, m, H-6), 2.43 (2H, m, H-5a and H-7a), 2.13
sodium chloride solution, dried with sodium sulfate, and then (4H, s, H-1 and H-3), 2.11 (2H, m, H-5b and H-7b); 13C-NMR
evaporated. The residue was purified by silica gel column (100MHz, CDCl3) δ: 166.6, 138.2, 133.1, 130.0, 129.6, 128.4,
chromatography (40% AcOEt in hexane) to give the ditosylate 128.4, 127.8, 127.6, 70.1, 68.5, 68.0, 67.9, 44.2, 39.6, 38.8,
as an oil (4.70g, 8.81mmol, 95%).
Next, disopropylmalonate (714.9µL, 3.76mmol) was slowly 493.19855. Found 493.19666.
37.3, 29.5; HR-MS (ESI) Calcd for C30H30NaO5 [M+Na]+:
added to a suspension of NaH (60% suspension in mineral oil)
2,2-Bis(benzoyloxymethyl)-6-hydroxyspiro[3.3]heptane
(106.9mg, 4.17mmol) in dry N,N-dimethylformamide (DMF) (12) To a solution of this compound 11 (194.7mg, 0.41mmol)
(10.0mL) under stirring at such a rate that the temperature in methanol (5.0mL) was added 10% Pd on carbon (136.8mg).
was maintained below 70°C. The ditosylate (1.00g, 1.88mmol) The reaction mixture was stirred at room temperature under
and KI (31.5mg, 0.19mmol) were added to the mixture in H2 atmosphere (1atm) for 10h. The suspension was filtered
one portion. The mixture was then stirred at 140°C for 14h. through a pad of Celite. The filtrate was evaporated, and then
Then, it was cooled, poured into saturated aqueous solution the residue was purified by silica gel column chromatography
of ammonium chloride, and the product was extracted with (60% AcOEt in hexane) to give a colorless oil 12 (112.8mg,
1
AcOEt, washed with saturated aqueous sodium chloride so- 0.30mmol, 72%). H-NMR (400MHz, CDCl3) δ: 8.02 (4H, m,
lution, dried with sodium sulfate, and then evaporated. The Bz), 7.55 (2H, m, Bz), 7.42 (4H, m, Bz), 4.40 and 4.41 (6H, s,
residue was purified by silica gel column chromatography 2-CH2OBz ×2 and Bn), 4.20 (1H, m, H-6), 2.49 (2H, m, H-5a
(25% AcOEt in hexane) to give as a colorless oil 9 (646.0mg, and H-7a), 2.12 and 2.14 (4H, s, H-1 and H-3), 2.02 (2H, m,
1.73mmol, 92%). 1H-NMR (400MHz, CDCl3) δ: 7.26–7.35 H-5b and H-7b); 13C-NMR (100MHz, CDCl3) δ: 166.6, 133.1,
i
(5H, m, Bn), 5.03 (2H, hept, J=6.0Hz, Pr), 4.37 (2H, s, Bn), 130.0, 129.6, 128.4, 68.0, 62.8, 47.1, 39.4, 38.6, 37.3, 28.7;
3.92 (1H, m, H-6), 2.53 (4H, s, H-1 and H-3), 2.36 (2H, m, HR-MS (ESI) Calcd for C23H24NaO5 [M+Na]+: 403.15159.
H-5a and H-7a), 2.02 (2H, m, H-5b and H-7b), 1.22 (12H, d, Found 403.15044.
i
J=6.0Hz, Pr); 13C-NMR (100MHz, CDCl3) δ: 171.3, 138.2,
2,6-Dichloro-9-[2′,2′-bis(benzoyloxymethyl)spiro[3′.3′]-
128.4, 127.8, 127.6, 70.0, 68.7, 68.2, 49.3, 43.0, 40.7, 30.4, 21.5; hept-6′-yl]purine (13) A solution of compound 12 (102.8mg,
HR-MS (ESI) Calcd for C22H30NaO5 [M+Na]+: 397.19855. 0.27mmol), triphenylphosphine (85.0mg, 0.32mmol), 2,6-di-
Found 397.19838.
chloropurine (61.3mg, 0.32mmol) and DIAD (diisopropyl

708
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
azodicarbosylate, 63.8µL, 0.32mmol) in THF (3.0mL) was test using STAT VIEW II (Abacus Concepts). A p value less
stirred at 50°C. After 20h stirring, the residual solution was than 0.05 was considered to be statistically significant.
purified by silica gel column chromatography (60% AcOEt
in hexane) to give as a colorless oil 13 (127.7mg, 0.23mmol,
Acknowledgment This research was partially supported
1
86%). H-NMR (400MHz, CDCl3) δ: 8.15 (1H, s, H-8), 8.04 by a Grant-in-Aid for Young Scientists (B), No. 24790123,
(4H, m, Bz), 7.58 (2H, m, Bz), 7.45 (4H, m, Bz), 4.98 (1H, from the Japan Society for the Promotion of Science (JSPS).
m, H-6′), 4.46 and 4.47 (4H, s, 2′-CH2OBz ×2), 2.86 (2H,
m, H-5′a and H-7′a), 2.73 (2H, m, H-5′b and H-7′b), 2.29 and
Conflict of Interest The authors declare no conflict of
2.38 (4H, s, H-1′ and H-3′); 13C-NMR (100MHz, CDCl3) δ: interest.
166.7, 152.9, 152.8, 151.8, 144.0, 133.3, 131.1, 129.8, 129.6,
128.5, 128.4, 67.7, 53.4, 43.5, 38.9, 38.3, 37.4, 31.6; HR-MS
(ESI) Calcd for C28H24Cl2N4NaO4 [M+Na]+: 573.10668. Found
573.10532.
References
1) Bobek V., Taltynov O., Pinterova D., Kolostova K., Vascul. Pharma-
col., 44, 395–405 (2006).
2) Collinson D. J., Donnelly R., Eur. J. Vasc. Endovasc. Surg., 28,
9–23 (2004).
3) O’Toole G., MacKenzie D., Buckley M. F., Lindeman R., Poole M.,
Br. J. Plast. Surg., 54, 1–7 (2001).
4) Tsukamoto I., Sakakibara N., Maruyama T., Igarashi J., Kosaka H.,
Kubota Y., Tokuda M., Ashino H., Hattori K., Tanaka S., Kawata
M., Konishi R., Biochem. Biophys. Res. Commun., 399, 699–704
(2010).
2-Amino-6-chloro-9-[2′,2′-bis(hydroxymethyl)spiro[3′.3′]-
hept-6′-yl]purine (2f) Compound 13 (122.6mg, 0.22mmol)
was dissolved in NH3 (14.0mL)/MeOH (3.0mL), and then
sealed and stirred for 1d at 100°C. The mixture was evaporat-
ed, and the residue was purified by silica gel column chroma-
tography (25% MeOH in CH2Cl2) to give 2f as white crystals
1
(50.3mg, 0.15mmol, 70%). H-NMR (400MHz, DMSO-d6) δ:
8.31 (1H, s, H-8), 7.72 (2H, brs, 6-NH2), 4.74 (1H, m, H-6′),
4.47 (2H, t, J=5.6Hz, 2′-CH2OH×2), 3.34 (4H, t, J=5.6Hz,
2′-CH2OH ×2), 2.48–2.60 (4H, m, H-5′ and H-7′), 1.84 and
1.96 (4H, s, H-1′ and H-3′); 13C-NMR (100MHz, DMSO-d6)
δ: 156.7, 152.7, 150.3, 139.9, 118.0, 65.0, 43.9, 43.2, 37.7, 37.0,
30.7; HR-MS (ESI) Calcd for C14H18ClN5NaO2 [M+Na]+:
346.10412. Found 346.10258; mp: 233.8°C.
5) Tsukamoto I., Konishi R., Tokuda M., Kubota Y., Maruyama
T., Kosaka H., Igarashi J., PCT Int. Appl., WO 2010061931 A1
20100603 (2010).
6) Sakakibara N., Tsukamoto I., Tsurura T., Takata M., Konishi R.,
Maruyama T., Heterocycles, 85, 1105–1116 (2012).
7) Okabe N., Nakamura E., Himi N., Narita K., Tsukamoto I.,
Maruyama T., Sakakibara N., Nakamura T., Itano T., Miyamoto O.,
Brain Res., 1506, 115–131 (2013).
Assay Angiogenesis Kit, Tubule Staining Kit for CD31,
human umbilical vein endothelial cells (HUVEC), fetal bovine
serum (FBS), vascular endothelial growth factor-A (VEGF),
HuMedia EG2, and HuMedia EB2 were purchased from
Kurabo Co. (Osaka, Japan). Cell Counting Kit 8 was supplied
by Dojindo Molecular Technologies (Kumamoto, Japan).
Cell Culture A co-culture system of HUVEC and human
fibroblasts (Angiogenesis Kit) was supplied in 24-well plates
by Kurabo.4) Cells were incubated for 10d prior to analysis
with 450µL of the culture medium and a 50µL of saline that
includes various additives. Culture medium was changed
every 3d, each time including freshly prepared additives.
Tube Formation Assay Ten days following incubation
periods with co-cultured fibroblasts and substrates (2a–f),
HUVEC were stained using Tubule Staining Kit for CD31.4)
The area of the formed tube was measured by the ImageJ
program. Two pictures from each well were provided for the
estimation. VEGF (10ng/mL) was used as a positive control.
Proliferation Assay4) HUVECs were seeded on gelatin-
coated 96-well plates, typically at 3000cells/well in 100µL
of maintenance medium. After seeding, the plates were incu-
bated for 24h to permit anchorage, and then, the culture me-
dium was changed with the assay medium, consisting of 90µL
of HuMedia EB2 with 2% heat-inactivated FBS and 10µL of
saline containing additives. The contents of HuMedia EB2
are almost identical to those of HuMedia EG2, except for the
fact that the former does not include FBS, a growth factor, or
antibiotics. The proliferation assay was performed using a Cell
Counting Kit-8 48h after the addition of compounds 2a–f.
Statistics All experiments were performed at least 5
times. Mean values and standard error (S.E.) of the mean are
shown. Statistical differences between groups were analyzed
by Student’s t-test and Dunnett’s multiple comparison test
using Microsoft Excel, and ANOVA followed by Scheffe’s F
8) Mendelson K., Evans T., Hla T., Development, 141, 5–9 (2014).
9) Igarashi J., Hashimoto T., Kubota Y., Shoji K., Maruyama T.,
Sakakibara N., Takuwa Y., Ujihara Y., Katanosaka Y., Mohri S.,
Naruse K., Yamashita T., Okamoto R., Hirano K., Kosaka H.,
Takata M., Konishi R., Tsukamoto I., Pharmacology Research &
Perspectives, 2, e00068 (2014).
10) Biswas K., Yoshioka K., Asanuma K., Okamoto Y., Takuwa N.,
Sasaki T., Takuwa Y., J. Biol. Chem., 288, 2325–2339 (2013).
11) Igarashi J., Miyoshi M., Hashimoto T., Kubota Y., Kosaka H., Am.
J. Physiol. Cell Physiol., 292, C740–C748 (2007).
12) Rikitake Y., Hirata K., Kawashima S., Ozaki M., Takahashi T.,
Ogawa W., Inoue N., Yokoyama M., Arterioscler. Thromb. Vasc.
Biol., 22, 108–114 (2002).
13) Buzard D. J., Thatte J., Lerner M., Edwards J., Jones R. M., Expert
Opinion Therapeutic Patents, 18, 1141–1159 (2008).
14) Crosignani S., Bombrun A., Covini D., Maio M., Marin D., Quat-
tropani A., Swinnen D., Simpson D., Sauer W., Françon B., Martin
T., Cambet Y., Nichols A., Martinou I., Burgat-Charvillon F., Riv-
ron D., Donini C., Schott O., Eligert V., Novo-Perez L., Vitte P.-A.,
Arrighi J.-F., Bioorg. Med. Chem. Lett., 20, 1516–1519 (2010).
15) Shimizu H., Takahashi M., Kaneko T., Murakami T., Hakamata
Y., Kudou S., Kishi T., Fukuchi K., Iwanami S., Kuriyama K.,
Yasue T., Enosawa S., Matsumoto K., Takeyoshi I., Morishita Y.,
Kobayashi E., Circulation, 111, 222–229 (2005).
16) Bolli M. H., Abele S., Binkert C., Bravo R., Buchmann S., Bur D.,
Gatfield J., Hess P., Kohl C., Mangold C., Mathys B., Menyhart K.,
Müller C., Nayler O., Scherz M., Schmidt G., Sippel V., Steiner B.,
Strasser D., Treiber A., Weller T., J. Med. Chem., 53, 4198–4211
(2010).
17) Matloubian M., Lo C. G., Cinamon G., Lesneski M. J., Xu Y.,
Brinkmann V., Allende M. L., Proia R. L., Cyster J. G., Nature
(London), 427, 355–360 (2004).
18) Wei S. H., Rosen H., Matheu M. P., Sanna M. G., Wang S.-K., Jo E.,
Wong C.-H., Parker I., Cahalan M. D., Nat. Immunol., 6, 1228–1235
(2005).
19) Slusarchyk W. A., Young M. G., Bisacchi G. S., Hockstein D. R.,

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
M. G., Zahler R., J. Med. Chem., 34, 1415–1421 (1991).
709
Zahler R., Tetrahedron Lett., 30, 6453–6456 (1989).
20) Singh J., Bisacchi G. S., Ahmad S., Godfrey J. D. Jr., Kissick T. P.,
Mitt T., Kocy O., Vu T., Papaioannou C. G., Wong M. K., Heikes
J. E., Zahler R., Mueller R. H., Org. Process Res. Dev., 2, 393–399
(1998).
21) Henlin J. M., Rink H., Spieser E., Baschang G., Helv. Chim. Acta,
75, 589–603 (1992).
22) Slusarchyk W. A., U.S. Patent, U.S. 5369098A 19941129 (1994).
23) Tokunaga, T., Takasaki, T., Yoshida, K., Nagata, R., PCT Int. Appl.,
WO 2007032371 A1 20070322 (2007).
24) Bisacchi G. S., Braitman A., Cianci C. W., Clark J. M., Field A. K.,
Hagen M. E., Hockstein D. R., Malley M. F., Mitt T., Slusarchyk W.
A., Sundeen J. E., Terry B. J., Tuomari A. V., Weaver E. R., Young
25) Sakakibara N., Hamasaki T., Baba M., Demizu Y., Kurihara M.,
Irie K., Iwai M., Asada E., Kato Y., Maruyama T., Bioorg. Med.
Chem., 21, 5900–5906 (2013).
26) Maruyama T., Sato Y., Horii T., Shiota H., Nitta K., Shirasaka T.,
Mitsuya H., Honjo M., Chem. Pharm. Bull., 38, 2719–2725 (1990).
27) Radchenko D. S., Grygorenko O. O., Komarov I. V., Tetrahedron
Asymmetry, 19, 2924–2930 (2008).
28) Yonesu K., Kawase Y., Inoue T., Takagi N., Tsuchida J., Takuwa Y.,
Kumakura S., Nara F., Biochem. Pharmacol., 77, 1011–1020 (2009).
29) Wong M. L. H., Prawira A., Kaye A. H., Hovens C. M., J. Clin.
Neurosci., 16, 1119–1130 (2009).