Study on the synthesis and PKA-I binding activities of 5-alkynyl tubercidin analogues

Article abstract of DOI:10.1016/S0968-0896(01)00341-8

5-Alkynyl tubercidin analogues were synthesized and their biologica activities were evaluated. It was found that protein kinase A could be activated by 5-alkynyl tubercidin (9a) and cAMP- binding ability to PKA-I was selectively inhibited by it. Molecular

Full text of DOI:10.1016/S0968-0896(01)00341-8

Bioorganic & Medicinal Chemistry 10 (2002) 907–912
Study on the Synthesis and PKA-I Binding Activities of
5-Alkynyl Tubercidin Analogues
Liangren Zhang, Yunlong Zhang, Xianghui Li and Lihe Zhang*
National Research Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100083, China
Received 13 July 2001; accepted 21 September 2001
Abstract—5-Alkynyl tubercidin analogues were synthesized and their biological activities were evaluated. It was found that protein
kinase A could be activated by 5-alkynyl tubercidin (9a) and cAMP-binding ability to PKA-I was selectively inhibited by it.
Molecular modeling showed that the interaction of 9a and PKA-I was associated with the existence of hydrophobic alkynyl group.
During the synthesis of tubercidin analogues, a pair of 20-carbonyl participating abnormal coupling products (11a, 11b) was
obtained, the structure was identified by X-ray crystalline diffraction. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cAMP analogues leads to inhibition of cancer cell
It was discovered that malfunctions of cellular signaling
have been associated with many diseases including can-
cer and diabetes.1À3 Many therapeutic strategies have
been developed through the design and synthesis of
compounds which activate or inactivate protein kinases.
Cyclic adenosine monophosphate (cAMP) dependent
pathway is one of the major signal transduction path-
ways in mammalian cells. cAMP is called second mes-
senger, it acts by binding to the regulatory subunits of
protein kinase A (PKA). PKA is a tetramer composed
of a regulatory subunits (R) dimer and two catalytic
(C) subunits. The regulatory subunits of isozyme I
and II (RI and RII, respectively) are dissimilar. RI
subunit overexpresses in cancer cell lines and tumor
specimens, decreasing RI/RII ratios and/or down-reg-
ulation of RI correlate with growth inhibition and cel-
lular differentiation.4À6
growth in a wide variety of cancer cell types in vitro and
in vivo. From amongst those cAMP analogues, 8-Cl-
cAMP, in particular, has proven strong antitumor
activity and currently is in clinical Phase II trials as an
anticancer agent, and it is suggested as a potential part-
ner for co-treatment with classical chemotherapeutics.4
Upon on the intensive investigation, it has found that
8-Cl-cAMP is relatively easy to be metabolized in vivo,
and the metabolite 8-Cl-adenosine also demonstrates
very active antitumor activity. Though the action mode
of 8-Cl-adenosine is not well understood, it is reported
to decrease the RI-subunit of PKA in lung epithe-
lial cells and also induce HL-60 promyelocytic
leukemia cells apoptosis.9,10 The biological activity
of 8-Cl-adenosine reveals that nucleoside might also
modulate protein kinase behavior as its cyclic
nucleotide does, and encourage us to find other
more active nucleoside which target the signal
pathway.
Regulations of signal transduction by synthetic mol-
ecules have been subjects of intense research in the
industrial setting as well as in academics. Several studies
have demonstrated that different antisense oligonucleo-
tides targeting the RIa subunit of PKA-I expression
causes cell growth arrest and differentiation in a variety
of cancer cell lines.7,8 It has also been shown that the
selective regulation of PKA-I or PKA-II by site selective
Tubercidin (7-deaza-adenosine) (1a) as well as 5-sub-
stituted derivatives—toyocamycin (1b) and sangivamy-
cin (1c)—are naturally occurring antibiotics (Scheme
1),11 their analogues have been synthesized in the past
years, some of them exhibited antitumor activities and
showed mild effect on PKA activity.12À15 In this study,
a hydrophobic alkynyl group was introduced to the
5-position of tubercidin and the designed compound 9
was synthesized and the biological activities were eval-
uated primarily.
*Corresponding author. Tel.: +86-10-6209-1700; fax: +86-10-6209-
2724; e-mail: zdszlh@mail.bjmu.edu.cn
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00341-8

908
L. Zhang et al. / Bioorg. Med. Chem. 10 (2002) 907–912
Results and Discussion
midine (6) and a-anomor was also generated (a/b 1:12).
6 was treated with CF3COOH and the deprotected
product 7 was obtained almost quantitatively. Amino-
lyzing 7 with saturated NH3/CH3OH, then alkynylating
with 1-hexyne or 1-octyne under the existence of
Pd(PPh3)4 and CuI, the alkynylated product 9a and 9b
were obtained in high yields. In another case, if 7 was
hydrolyzed in methanol in the presence of K2CO3,
compound 8b was formed, the alkynylated product 9c
and 9d could be obtained (Scheme 2).
Synthesis
5-alkynyl tubercidin analogues (9) were synthesized via
the routine as demonstrated in Scheme 2. 4-Chloro-5-
iodo-7H-pyrrolo[2,3-d]pyrimidine (3) reacted with
freshly prepared 1-chloro-5-O-tert-butyldimethylsilyl-
2,3-O-isopropylidene-a-d-ribofuranose (5) to give 4-
chloro-5-iodo-(50-O-tert-butyldimethylsilyl-20,30-O-iso-
propylilene-b-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
For the synthesis of compound 7, protected a-d-ribosyl
chloride 5 was coupled with 4-chloro-7H-pyrrolo[2,3-
d]pyrimidine (2) followed by iodolization with ICl.
Unfortunately, a mixture of 6 and 6a was generated
(Scheme 3). The result indicated that the ribosyl group
affected the nucleophilicity of C-5 and C-6 in tubercidin
analogue.
When using acetyl as the protecting group in b-d-ribo-
furanosyl chloride (10) for the coupling reaction with
4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2), an abnormal
Scheme 1.
Scheme 3. (i) NaH, MeCN, rt, 24 h; (ii) ICI, rt, 72 h.
Scheme 2. (i) NaH, rt, 20 h; (ii) CCI4, P(NMe2)3, À78 ꢀC, 1 h; (iii)
NaH, rt, 24 h; (iv) CF3COOH, rt, 1 h; (v) NH3/CH3OH, 110 ꢀC, 22 h;
(vi) K2CO3/MeOH, rt, 12 h; (vii) Pd(Ph3)4, Cul, DMF, rt, 16 h.
Scheme 4. (i) NaH, CH2CI2, rt, 48 h.

L. Zhang et al. / Bioorg. Med. Chem. 10 (2002) 907–912
909
coupling reaction was observed (Scheme 4). 10 was
reacted with 2 in dichloromethane at room temperature
under the existence of NaH, two compounds were
obtained with the yield of 41% (11a) and 21% (11b),
respectively. NMR results demonstrated that neither
11a nor 11b were consistent with the desired product.
13C NMR showed that only two carbonyl groups exis-
ted in both compounds and a new peak appeared at d
114.9 (11a) or 115.8 (11b). HMBC and HMQC identi-
fied that this carbon was connected to a methyl group.
After 11a or 11b was treated with saturated ammonia
methanol solution, two signals of carbonyl groups dis-
appeared in 13C NMR but signal of 114.9 or 115.8 still
existed in the deprotected product. All spectrometric
data of 11a or 11b were consistent with the structure in
which the heterocyclic base was connected to the carbon
of 1,2-O-ethylidene moiety of 3,5-diacetyl-ribose. A
crystalline sample of 11a was obtained from petroleum–
dichloromethane solution, and its X-ray diffraction
results confirmed the structure and showed that new
chiral center, C-7 of 11a, was in S configuration (Fig. 1).
Since the spectrometric data of 11b was nearly identical
to 11a, it should be a stereo isomer of 11a with R con-
figuration of its C-7.
cyclic bases were used as reactants. Similar coupling
products were reported by Cristalli and Revankar.16,17
De Clercq also proposed an abnormal coupling struc-
ture in 1995, in which an O-5 carbonyl other than O-2
carbonyl participating product was suggested.18 Crystal
structure presented in this work clarified the O-2 car-
bonyl neighboring participation mechanism.
Biological evaluation
Compounds 9a–d and 11a–b were screened by culture of
tumor cells, 9a and 9b exhibited relatively high inhibi-
tion effects on the proliferation of KB, Hela and Bel-
7402 cells (Table 1), otherwise, 9c, 9d, 11a and 11b did
not show obvious effects on cell growth. It indicated
that the tubercidin substituted skeleton and the 4-amino
group was essential to the inhibition activities of tumor
cell growth.
The effects on protein kinase A activity by compound
9a in the concentration range of 0.1–10 mM was inves-
tigated (Fig. 3). The results showed that PKA was acti-
vated by 9a, which was in accordance with most cyclic-
AMP derivatives and 8-Cl-adenosine, it seemed that 9a
acted in the similar way as they did. Whereas it was
reported that toyocamycin and tubercidin inhibited
PKA activity with IC50 of 20 and >100 mM, respec-
tively,14 it indicated that 9a and tubercidin affected
PKA activity in a different way. The V-shaped variation
of PKA activities might be resulted from the binding
differences of 9a to PKA-I and PKA-II. The selective
binding ability of compound 9a to PKA was examined
by competitive cAMP-[3H] binding assay (Fig. 4). The
result showed that 9a could inhibit cAMP binding to
PKA-I very effectively and exhibited concentration
dependence, meanwhile no obvious difference to control
was observed in PKA-II case. It might be concluded
that the alkynyl tubercidin could target to PKA-I selec-
tively.
The formation of 11a and 11b could be explained by the
neighboring participation effect of acetyl group. The
heterocyclic base attacked the carbon of the cation
intermediate (path a) instead of the ribosyl C-1 carbon
(path b) (Fig. 2). This abnormal coupling reactions were
occasionally observed when weak nucleophilic hetero-
Table 1. Growth inhibition of tubercidin and its derivatives to
various tumor cells
Compound
IC50 (mM)
KB
Hela
Bel-7402
9a
9b
0.3
0.1
1.8
0.4
0.2
0.45
0.55
0.25
0.25
Figure 1. ORTEP drawing of X-ray diffraction structure of compound
11a.
Tubercidin
Figure 2. Schematic representation of neighboring acetic group parti-
cipating effect on formation of normal (path a) and abnormal (path b)
products.
Figure 3. Effect of compound 9a on PKA activity (p<0.05).

910
L. Zhang et al. / Bioorg. Med. Chem. 10 (2002) 907–912
For understanding the mechanism more intensively,
molecular modeling was used to simulate the interaction
of 5-alkynyl tubercidin with PKA. Three-dimensional
structure of the regulatory subuint RIa of PKA clearly
showed that in the cAMP binding site there was a rela-
tively large hydrophobic area next to N-7 position of
cAMP. If the cAMP was replaced by 5-alkynyl tuberci-
din, the alkynyl group could fit this pocket tightly by the
hydrophobic interactions with the adjacent amino acid
residues, such as Leu 112 (Fig. 5). It implied that the
hydrophobic substitution at 5-position of tubercidin
could be contributed to the selective binding activity to
PKA-I. It would be interesting to provide a strategy for
the development of new antitumor candidates from the
structural modification of tubercidin.
4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (3) was
synthesized according to refs 19 and 20. 4-Chloro-5-iodo-
(b-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (7) was
synthesized according to references starting from the
coupling of 5-O-tert-butyldimethylsilyl-2,3-O-isopropy-
lidene-d-ribofuranose (4) and compound 3.12,21 PKA
assay kit (Spinzyme formate) was purchased from
Pierce. cAMP-[3H] assay kit was purchased from the
Institute of Atomic Power of China.
NMR spectra were recorded on Varian 300 MHz spec-
trometer. Mass spectra were obtained on ZAB-HS and
HRMS were recorded on APEX II FTICR (Bruker)
mass spectrometer. Optical rotation was measured on
Perkin-Elmer 243B polarimeter. Elemental analyses
were performed on PE-240C analyzer.
Experimental
Molecular modeling
Materials and methods
Molecular modeling was performed on SGI Indy work-
station and MSI Insight II suite was used. Three-
dimensional structure of PKA-I Ra subunit was from
Weber’s theoretical model (Protein Data Bank entry
1APK).22 Tubercidin derivative was placed to PKA-I
Ra cAMP binding site by modifying cAMP structure,
and was subjected to molecular mechanics energy mini-
mization (1000 steps steepest descent and 1000 steps
conjugate gradient) consequently. In the computation,
the amino acid residues which were 5 A away from
tubercidin derivative were fixed and others were unre-
strained, and the cvff forcefield was used.
d-Ribose, 1-hexyne, 1-octyne, Pd(PPh3)4 and other
main materials were purchased from ACROS. CH3CN,
pyridine, CH2Cl2, DMF were dried by distillation from
CaH2. Chromatographic purifications were carried out
using silica gel (200–400 mesh, Qinghdao Chemicals).
4-Amino-5-iodo-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (8a). Compound 7 (475 mg, 1.15 mmol) was
dissolved in 50 mL of saturated NH3/CH3OH solution.
After stirred at 116 ꢀC in a sealed vessel for 22 h, the
mixture was evaporated and separated with silica gel
column. 400 mg (yield 88.7%) of oil-like product was
collected. MS/EI: 391 (MÀ1). 1H NMR (DMSO-d6,
ppm). 8.09 (s, 1H, H-2); 7.66 (s, 1H,0H-6); 6.68 (s, 2H,
Figure 4. Effect of compound 9a on cAMP binding with PKA (blank),
PKA-I (cross) and PKA-II (gray) (p<0.05).
0
0
NH2); 6.01 (d, 1H, J1 ,2 =6.0 Hz, H-1 ); 4.34 (m, 1H, H-
20); 4.03 (m, 1H, H-30); (m, 1H, H-40); 3.57 (m, 2H, H-
50).
4-Methoxy-5-iodo-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (8b). Compound 7 (440 mg, 1.0 mmol) was
dissolved in 12 mL of CH3OH. 300 mg (2.0 mmol) of
K2CO3 was added and stirred at room temperature for
12 h. The mixture was evaporated and separated with
silica gel column. 250 mg (yield 88.7%) of solid product
was collected. Mp 212–213 ꢀC (dec). MS/EI: 407 (M).
1H NMR (DMSO-d6, ppm). 8.44 (s, 1H, H-2); 7.88 (s,
0
0
0
1H, H-6); 6.13 (d, 1H, J1 ,2 =6.0 Hz, H1 ); 4.37 (m, 1H,
H-20); 4.05 (m, 4H, H-30, OCH3); 3.95 (m, 1H, H-40);
3.57 (m, 2H, H-50).
General procedure for the synthesis of 4-amino-5-alky-
nyl-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo [2,3-d] pyrimidine
(9). 0.57 mmol of compound 7 was dissolved in 8 mL
of anhydrous DMF, 5.7 mmol of 1-hexyne or 1-octyne,
0.16 mL (1.15 mmol) of triethyl amine and 75 mg (0.065
mmol) of Pd(PPh3)4 were added under Ar atmosphere.
After the solution was stirred at room temperature for
Figure 5. Molecular modeling showing the interaction of compound
9a with amino acid residues of PKA-Ia.

L. Zhang et al. / Bioorg. Med. Chem. 10 (2002) 907–912
911
0
0
16 h, the mixture was evaporated under reduced pres-
sure. MeOH was added to dissolve residue and inso-
luble material was removed by filtration. Filtrate was
evaporated and separated with silica gel column, the
desired product was collected.
6.66 (d, 1H, J5,6=2.8 Hz. H-5); 6.21 (d, 1H, J1 ,2 =4.2
Hz, H-10); 4.93 (m, 1H, H-20); 4.83 (m, 1H, H-30); 4.33
(m, 2H, H-40, H-50); 4.13 (m, 1H, H-50); 2.22 (s, 3H,
CH3); 2.11 (s, 3H, CH3); 0.98 (s, 3H, CH3). 13C NMR
(DMSO-d6, ppm): 170.1, 169.6, 150.9, 150.4, 149.4,
128.1, 118.4, 114.9, 105.2, 98.6, 78.0, 76.0, 62.0, 24.6,
20.5, 20.4. Anal. calcd for C17H18ClN3O7: C 49.58, H
4.41, N 10.20; Found: C 50.00, H 4.44, N 10.26.
19
Compound 9a. Yield 56.8%. Mp 214–215 ꢀC (dec). ½ꢀꢁD
(c 0.165, MeOH) À49.6. UV (MeOH): lmax 277 nm (e
1
11,000). H NMR (CD3OD, ppm). 8.07 (s, 1H, H-2);
19
0
0
0
7.48 (s, 1H, H-6); 5.97 (d, 1H, J1 ,2 =6.3 Hz, H-1 ); 4.57
(m, 1H, H-20); 4.26 (m, 1H, H-30); 4.09 (m, 1H, H-40);
3.70 (m, 2H H-50); 2.48 (t, 2H, J=6.6 Hz, CCCH2);
1.63–1.46 (m, 4H, CH2); 0.97 (t, 3H, J=7.2 Hz, CH3).
HRMS calcd for C17H22N4O4 (M+1) 347.1713, found
347.1708.
Compound 11b. Syrup. ½ꢀꢁD (c 0.210, MeOH) +109.5.
UV (MeOH): lmax 269 nm (e 4200). 1H NMR
(300 MHz, DMSO-d6, ppm): 8.73 (s, 1H, H-2); 7.78 (d,
1H, J6,5=3.9 Hz, H-6); 6.71 (d, 1H, J5,6=3.6 Hz, H-5);
0
0
0
0
6.24 (d, 1H, J1 ,2 =4.2 Hz, H-1 ); 5.18 (m, 1H, H-2 );
4.68 (m, 1H, H-30); 3.95 (m, 3H, H-40, H-50); 2.00 (s, 3H,
CH3); 1.98 (s, 3H, CH3); 1.73 (s, 3H, CH3). 13C NMR
(DMSO-d6, ppm): 169.9, 169.3, 150.9, 150.6, 149.8,
127.8, 118.3, 115.8, 106.3, 98.5, 79.2, 77.9, 71.7, 62.8,
26.4, 20.5, 19.9. HRMS calcd for C17H18ClN3O7
(M+H) 412.0901, found 412.0898.
19
Compound 9b. Yield 50.8%. Syrup. ½ꢀꢁD (c 0.190,
1
MeOH) À62.1. UV (MeOH): lmax 276 nm (e 7700). H
NMR (CD3OD, ppm). 8.05 (s, 1H, H-2); 7.47 (s, 1H, H-
0
0
0
0
6); 5.97 (d, 1H, J1 ,2 =6.3 Hz, H-1 ); 4.56 (m, 1H, H-2 );
4.25 (m, 1H, H-30); 4.08 (m, 1H, H-40); 3.81 (m, 2H, H-
50); 2.45 (t, 2H, J=6.9 Hz, CCCH2); 1.62–1.30 (m, 4H,
CH2); 0.91 (t, 3H, J=6.9 Hz, CH3). HRMS calcd for
C19H26N4O4 (M+1) 375.2026, found 375.2022.
Measurement of biological behaviors
The inhibition of tumor cell growth was measured by
SRB assay. PKA activity was measured using the col-
orimetric PKA assay kit.23 The treated cell were washed
with ice-cold PBS three times, resuspended in lysis buf-
fer (50 mM Tris–HCl, 5 mM EDTA, 10 mM NaF, 10%
glycerol, pH 7.2), sonicated, and centrifuged. Super-
natants then were taken as lysate. PKA activity assays
were performed following the manufacturer’s instruc-
tions included with the colorimetric PKA assay kit. The
cAMP contents were measured using the cAMP-[3H]
assay kit according to the manufacturer’s instruction.
PKA-I and PKA-II were separated with reference’s
method.24,25
19
Compound 9c. Yield 54.3%. Mp 123–125 ꢀC (dec). ½ꢀꢁD
(c 0.245, MeOH) À61.1. UV (MeOH): lmax 272 nm (e
1
7500). MS/EI: 361 (M). H NMR (CD3OD, ppm). 8.44
0
0
(s, 1H, H-2); 7.87 (s, 1H, H-6); 6.12 (d, 1H, J1 ,2 =6.0
Hz, H-10); 4.36 (m, 1H, H-20); 4.07 (m, 4H, H-30,
OCH3); 3.90 (m, 1H, H-40); 3.63 (m, 2H, H-50); 2.44 (t,
2H, J=6.6 Hz, CCCH2); 1.50 (m, 4H, CH2); 0.94 (t,
3H, J=6.6 Hz CH3). Anal. calcd for C18H23N3O5: C
59.82, H 6.41, N 11.63; found C 59.68, H 6.49, N 11.47.
19
Compound 9d. Yield 46.0%. Mp 119–123 ꢀC (dec). ½ꢀꢁD
(c 0.190, MeOH) À48.4. UV (MeOH): lmax 268 nm (e
1
7500). MS/EI: 389 (M). H NMR (CD3OD, ppm). 8.44
0
0
(s, 1H, H-2); 7.86 (s, 1H, H-6); 6.12 (d, 1H, J1 ,2 =6.0
Hz, H-10); 4.22 (m, 1H, H-20); 4.09 (m, 4H, H-30,
OCH3); 3.91 (m, 1H, H-40); 3.56 (m, 2H, H-50); 2.43 (t,
2H, J=6.6 Hz, CCCH2); 1.55–1.31 (m, 8H, CH2); 0.89
(t, 3H, J=6.6 Hz, CH3). Anal. calcd for C20H27N3O5: C
61.68, H 6.99, N 10.79; found C 61.79, H 7.14, N 10.58.
Acknowledgement
The research was supported by the National Natural
Science Foundation of China.
3,5-Di-O-acetyl-1,2-O-(methyl-1-(4-chloro-pyrrolo[2,3-d]-
pyrimidin-7-yl) methylene)-ꢁ-D-funarose (11). 31 mg
(0.2 mmol) of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was
suspended in 5 mL of CH2Cl2, then 46 mg (1.0 mmol) of
52% NaH was added. After stirred at room temperature
for 30 min, 0.32 g (1 mmol) of 1-chloro-2,3,4-tri-O-b-d-
ribose (9) (dissolved in 5 mL of CH2Cl2) was added and
stirred at room temperature for 48 h. After evaporation,
ethyl acetate was used to extract the residue for three
times (5 mL each). Ethyl acetate solution was combined
and separated with silica gel chromatography, 34 mg of
11a (yield 41.0%) and 17 mg of 11b (yield 20.5%) were
collected, respectively.
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19
Compound 11a. Mp 115–117 ꢀC (dec). ½ꢀꢁD (c 0.175,
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MS/FAB: 412 (M+H). 1H NMR (300 MHz, DMSO-d6,
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