Synthesis and biological evaluation of novel homocamptothecins conjugating with dihydropyrimidine derivatives as potent topoisomerase i inhibitors

Article abstract of DOI:10.1002/ardp.201000402

Homocamptothecin (hCPT) is a camptothecin (CPT) homologue with the insertion of a methylene (-CH₂-) spacer between the alcohol moiety and carbonyl group of the classical six-membered α-hydroxylactone ring. This modification provides higher lact

Full text of DOI:10.1002/ardp.201000402

726
Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Full Paper
Synthesis and Biological Evaluation of Novel
Homocamptothecins Conjugating with Dihydropyrimidine
Derivatives as Potent Topoisomerase I Inhibitors
Lingjian Zhu1, Pengfei Cheng1, Ning Lei2, Jianzhong Yao1, Chunquan Sheng1, Chunlin Zhuang1,
Wei Guo1, Wenfeng Liu1, Yongqiang Zhang1, Guoqiang Dong1, Shengzhang Wang1,
Zhenyuan Miao1, and Wannian Zhang1
1 School of Pharmacy, Second Military Medical University, Shanghai, P. R. China
2 Department of Quality Assurance, Lian Yun Gang JARI Pharmaceutical Co., Ltd., Lianyungang, P. R. China
Homocamptothecin (hCPT) is a camptothecin (CPT) homologue with the insertion of a methylene
(–CH2–) spacer between the alcohol moiety and carbonyl group of the classical six-membered
a-hydroxylactone ring. This modification provides higher lactone stability and did not impair its
activity against topoisomerase I (Topo I), but rather appears to improve it compared to CPT. In an
attempt to improve the antitumor activity of homocamptothecins, a series of novel hCPT derivatives
conjugating with dihydropyrimidine (DHPM) derivatives was designed and synthesized based on a
synthetic route which couples 7-formylhomocamptothecin with different dihydropyrimidine
derivates. Most of the synthesized compounds exhibited good antiproliferative activity on tumor
cell lines A549, MDA-MB-435 and HCT116. Furthermore, this class of compounds showed superior
Topo I inhibition activity comparable to or higher than CPT.
Keywords: Biological activity / Camptothecin / Dihydropyrimidine / Homocamptothecin
Received: December 28, 2010; Revised: February 28, 2011; Accepted: March 2, 2011
DOI 10.1002/ardp.201000402
Introduction
class of compounds. The most important discovery would be
to modify the metabolically liable camptothecin lactone ring
from a six-membered a-hydroxylactone to a seven-membered
b-hydroxylactone ring, which leads to the development of the
homocamptothecin (hCPT) family of compounds [5].
Homocamptothecin represents a promising prototype for
the next generation of topoisomerase I targeting agents.
Upgrading the highly reactive six-membered lactone to a
more stable seven-membered ring is achieved without com-
promising any antitumor activity: hCPT not only retains
superior antitumor activity, it also has a reinforced capacity
to inhibit topoisomerase I compared with CPT [6]. According
to these encouraging results, many hCPT analogues have
been synthesized and evaluated in vitro and in vivo as the
antitumor agents [7–9]. Among them, diflomotecan
(BN80915) and elomotecan (BN80927) are representative ones
and have been in clinical trial, and encouraging results have
been obtained (Fig. 2) [10, 11]. Furthermore, a series of novel
homocamptothecin derivatives have been synthesized in our
(20S)-Camptothecin (CPT) [1], a cytotoxic quinoline alkaloid
which inhibits topoisomerase I (Topo I), is a potent anticancer
agent that was isolated from the Chinese tree Camptotheca
acumunata by Wani and Wall in 1966. It has already been
discovered that the cytotoxicity of camptothecin is due to a
novel mechanism of action involving selective inhibition of
DNA topoisomerase I (Topo I) [2, 3]. Its two water-soluble
analogs, topotecan and irinotecan, are used in clinics as
anticancer agents (Fig. 1).
However, the severe toxicity, the instability of the lactone
and other defects of CPTs [4] have promoted intensive efforts
to increase the efficacy of these agents, reduce their toxicity
in patients, and overcome the tumor drug resistance for this
Correspondence: W. N. Zhang, Z. Y. Miao, C. Q. Sheng, School of
Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai
200433, P. R. China.
E-mail: zhangwnk@hotmail.com; miaozhenyuan@hotmail.com;
shengcq@hotmail.com
Fax: þ86-21-81871243
These authors contributed equally to this work:
L.-J. Zhu, lj_zhu@163.com; P.-F. Cheng, chengpf@163.com
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Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Novel Homocamptothecins Conjugating with Dihydropyrimidine Derivatives
727
R2 R1
camptothecin
R1=R2=R3=H
R3
O
N
D
A
B
N
C
irinotecan
OCON
N
R1=Et, R2=H, R3=
O
E
topotecan
R1=H, R2=CH2NMe2, R3=OH
OH
O
Figure 1. Camptothecin and representative analogs.
R1
9
7
10
R2
R3
=
=
=
homocamptothecin
diflomotecan
R1 R2 R3 H
O
N
N
=
=
=
R1 H R R3 F
,
2
O
elomotecan
=
e
M
,
=
R
Cl
3
O
R2
.
C
H
HCl
3
=
R1
H
2C
N
H
O
Figure 2. Homocamptothecin and representative analogs.
laboratory and several compounds that showed superior
activity had been chosen to conduct preclinical studies
[12–14]. Such findings encouraged us to develop novel and
semisynthetic analogues of hCPT with improved pharmaco-
logical and pharmaceutical properties while maintaining
(or even increasing) the antitumor activity of the parent
compound.
Figure 3. Hypothetical model for the binding of homocamptothecin-
DHP conjugate (4h) with DNA–Topo I complex. The figure was
prepared from PDB entry: 1T8I, using PyMol (http://pymol.
souceforge.net/).
For above-mentioned pharmacological potency of the DHPM
scaffold, we were interested in studying the incorporation of
a dihydropyrimidine unit into the homocamptothecin
skeleton in order that we could discover novel hCPT
analogues with higher Topo I inhibition efficacy, better thera-
peutic effect and lower toxicity. Although the volume of
dihydropyrimidine derivatives is relatively big, based on
the ternary complex of DNA–Topo I-CPT [27], we presented
a reasonable binding mode of this class of compounds with
DNA–Topo I complex. In this model, we found a large space
around the C-7 position of hCPT that allowed the introduc-
tion of DHPM derivatives, and two key hydrogen bonds were
reserved (Fig. 3).
The high stability of hCPT is with no doubt directly attrib-
uted to its expanded lactone E-ring but the activity and to-
poisomerase I inhibition activity can be enhanced further by
adding a substituent to the native scaffold. In recent years,
the use of conjugates has emerged as a frequent strategy in
efforts to optimize therapeutically beneficial properties of
CPT, including lactone stability, solubility/lipophilicity,
tumor cell recognition and sequence specificity of DNA dam-
age [15–18]. Dihydropyrimidine (DHPM) and their derivatives
of the Biginelli-type have occupied an important place in
natural and synthetic organic chemistry mainly due to their
wide range of biological activities [19], notably as calcium
channel blockers [20]. Additionally, their particular structure
has been found in natural marine alkaloid batzalladines Result and discussion
which are the first low molecular weight natural products
reported in the literature to inhibit the binding of HIV gp-120
to CD4þ cell, so disclosing new vistas towards the develop-
ment of AIDS therapy [21, 22]. Meaningfully, the structurally
related derivative monastrol was identified as a novel low
molecular weight cell-permeable molecule for the develop-
ment of potentially new anticancer drugs [23], and several
screening assays indicated that this type of compounds exhib-
ited effective antiproliferative activity against several cancer
cell lines [24]. Furthermore, the past researches revealed that
there was a potential correlation between cancer and hyper-
tension which is actually a significant risk factor for cancer
[25, 26]. Therefore, we supposed that introducing the organic
molecules with hypotensive effect into antitumor molecules
may be an effective measure to improve therapeutic effect of
antitumor agents.
Chemistry
To test the hypothesis concerning the effect of DHPM deriva-
tives on position 7 of hCPT, we prepared twenty hCPT-DHPM
derivatives in racemic mixtures. The chemical synthesis of
the targeted compounds was based on two key intermediates,
dihydropyrimidine derivatives and 7-formylhomocampto-
thecin. We employed classical Biginelli reaction [28] to syn-
thesize the nitro substituted DHPM derivatives 2a–j, then
reduced to get amino substituted DHPM derivatives 3a–j
(Scheme 1). 7-Formylhomocamptothecin was prepared from
hCPT according to the method reported by Sawada et al. [29].
Due to the acetoxymethyl hCPT as the main by-product, SeO2
was used as the oxidant instead of previous acetic acid in this
reaction (Scheme 2). Scheme 3 shows the synthetic strategy
for our targeted compounds 4a–t based on the coupling of
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L.-J. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
O
R2
N OH
O
R2
N OH
HN
CF3
HN
CF3
CHO
O
O
O
i
R2
ii
+
+
R1
R1
H2N
N
H
F3C
R1
O
O
NO2
NO2
2a-e
NH2
1a p
1b m
3a-e
O
R2
O
R2
N
N
CHO
O
O
O
HN
CH3
R1
HN
CH3
R1
i
R2
ii
+
+
H3C
R1
H2N
N
H
NO2
O
O
NO2
2f-t
NH2
1a p
1b m
3f-t
Scheme 1. Reagents and conditions: (i) TMSCl, DMF, r.t.; (ii) H2/Pd-C, EtOH, r.t.
H
O
H
C
O
O
O
N
O
i
ii
N
N
N
Scheme 2. Reagents and conditions: (i)
75% H2SO4, FeSO4 ꢀ 7 H2O, 30% H2O2,
CH3OH, H2O, 12h, 79%; (ii) SeO2/dioxane,
1008C, 8 h, 85%.
N
O
N
O
O
O
H
O
O
O
H
O
H
O
various DHPM derivatives and 7-formylhomocamptothecin.
–
respectively. Importantly, compared with hCPT, most of the
synthesized compounds showed enhanced antitumor
activity regardless of whether against A549, MDA-MB-435
and HCT116 cell lines. The bulky substitution conferred by
DHPM and the diverse pharmacological properties of DHPM
are the two main considerations for our original design idea,
which was well proved by the research results. Furthermore,
four compounds, 4b, 4h, 4k, and 4m, possessed high growth
inhibitory effect at the concentration of 0.01, 0.1, 1, 10, and
100 mg/mL, while relatively low activity of TPT was observed
at the same concentration (Fig. 4). In particular, the activities
of the most promising compounds, 4b and 4m, showed broad
in-vitro antitumor spectrum.
The bond C N was selected as a linker, which was success-
–
fully adopted in our previous drug design [13].
In-vitro antitumor activity
The in-vitro antitumor activities of these DHPM-hCPT deriva-
tives against solid tumor cell lines, A549 (for non-small cell
lung cancer), MDA-MB-435 (for breast cancer), and HCT116
(for colon cancer), were evaluated. Topotecan was used as a
positive compound since it is a clinically effective CPT and
does not require metabolic activation.
Table 1 indicated that these DHPM derivatives on position
7 of hCPT could promote antitumor activity of hCPTs as
expected, which indicates the advantages of this strategy.
The results also revealed that these compounds were more
sensitive against MDA-MB-435 and HCT116 cell lines than
against A549 cell line. For A549 cell line, most of the tested
compounds showed increased cytotoxic potency compared
with TPT. With regard to MDA-MB-435 cell line, with few
exceptions (4e, 4q, and 4s), the tested compounds exhibited
higher antitumor activities than TPT. According to HCT116
cell line, compounds 4a–4o also showed higher in-vitro inhibi-
tory activities than TPT. Especially, the IC50 values of com-
pounds, 4b, 4k, and 4m, reached to 78 nM, 48 nM, and 81 nM,
Topoisomerase I inhibition activity assays
Generally, camptothecin and its clinically used derivatives
act by stabilizing a covalent topoisomerase I–DNA complex
called the cleavable complex. Therefore, topoisomerase I-
mediated DNA cleavage assay was used to investigate the
ability of the prepared compounds to inhibit Topo I.
Cleavable complexes were revealed by the appearance of
short DNA fragments when the samples were analyzed by
gel electrophoresis under denaturing conditions. The pre-
sented results (Fig. 5) indicated that compounds 4b, 4h, 4k,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Novel Homocamptothecins Conjugating with Dihydropyrimidine Derivatives
729
O
R2
N
O
R2
N OH
HN
CH3
R1
HN
CF3
R1
O
O
CHO
N
N
O
i
CH
CH
or
N
3a-t
+
O
N
O
N
N
O
N
N
O
O
O
OH
O
O
OH
OH
4f-t
R2
4a-e
R1
R2
R2
H
R1
R1
4a
4b
4c
4h
4i
p
p
4o
p
p
p
p
p
CH3CH2O
CH3CH2O
C2H5
CH3O
H
H
CH3O
m
p
H
4p
4q
4r
H3C
H3C
H3C
C2H5
H3C
C2H5
H3C
CH3CH2O
4j
p
p
CH3CH2O
H3C
(CH3)3CO
p
CH3CH2O
(CH3)2CHCO
(CH3)3CO
H3C
CH3CH2O
CH3CH2O
4d
4e
Ph
4k
4l
p
p
p
p
p
p
4s
C2H5
Ph
H
CH3CH2O
(CH3)3CO
Ph
p
C2H5
4f
4m
4n
4t
CH3CH2O
CH3CH2O
H
H
(CH3)2CHCO
4g
m
H
Scheme 3. Reagents and conditions: (i) Yb(OTf)3, CHCl3, r.t.
4l, 4m, 4n, and 4t as cytotoxic agents were also very potent as
topoisomerase I inhibitors. The results also showed that the
addition of increasing concentrations of CPT, 4b, or 4m is
accompanied by a dose-dependent increase in the level of
cleavable complexes. Importantly, even in low concentration
(1 mM), 4b and 4m showed obvious Topo I inhibition activity,
while CPT exhibited no inhibition activity (Fig. 6).
DHPM conjugates as effective cytotoxic agents also showed
superior Topo I inhibition activity. A better understanding of
this feature could provide meaningful insights for the develop-
ment of more effective topoisomerase I inhibitors.
Experimental
Chemistry
All reagents and solvents were reagent grade or were purified by
standard methods before use. The melting points were deter-
mined using an electrothermal apparatus and are uncorrected.
1H- and 13C-NMR spectra were recorded at 500 MHz with a Bruker
instrument, and reported with TMS as internal standard and
CDCl3 or DMSO-d6 as solvent. Chemical shifts (d values) and
coupling constants (J values) are given in ppm and Hz, respect-
ively. Elemental analyses were performed with a MOD-1106
instrument and were consistent with theoretical values within
ꢁ0.4%. TLC analysis was carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China). Flash column chromatog-
raphy was carried out on silica gel 200–300 mesh. Anhydrous
solvent and reagents were all analytically pure and dried
through routine protocols. All reactions requiring anhydrous
conditions were performed under a positive nitrogen flow, and
all glassware was oven-dried and/or flame-dried.
Conclusion
In conclusion, twenty hCPT-DHPM conjugates were synthes-
ized based on a semi-synthetic route. Most of the synthesized
compounds exhibited good antiproliferative activity on tumor
cell lines A549, MDA-MB-435 and HCT116. Especially, com-
pounds 4b, 4k, and 4m showed higher in-vitro inhibitory activi-
ties against HCT116 than TPT with the IC50 of 78 nM, 48 nM
and 81 nM, respectively. It is likely that the improvement of
cytotoxic activity is the result of multiple favorable events,
including DHPM’s biological diversity and increased drug
uptake (as expected on the basis of increased lipophilicity).
The results presented in this study also indicate that hCPT-
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L.-J. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Table 1. In-vitro antitumor activity of hCPT–DHPM derivative
conjugates a
Compounds
IC50 (mM)
A549
MDA-MB-435
HCT116
Figure 6. Effect of compounds CPT, 4b, and 4m on
Topoisomerase-I mediated DNA relaxation in concentration
gradient. Topo I was topoisomerase I and DNA without drugs; the
samples were reacted with 1, 50, and 100 mM drugs at 378C for
15 min. Reactions were then stopped by adding 0.5% SDS. Gels
were photographed under a UV transilluminator.
4a
4b
4c
4d
4e
1.905
0.891
2.469
1.798
3.935
1.808
1.947
1.974
2.236
1.837
1.770
1.618
0.158
2.162
4.099
6.434
2.389
1.988
3.821
1.860
2.980
3.159
0.328
0.365
0.0795
0.213
1.550
0.695
0.420
0.270
0.452
0.450
0.210
0.374
0.349
0.164
0.505
0.879
1.129
0.306
1.384
0.167
1.035
1.10
0.371
0.0785
0.155
0.148
0.244
0.200
0.310
0.206
0.307
0.240
0.0488
0.215
0.0813
0.160
0.258
0.977
1.398
0.940
1.984
0.741
0.944
0.448
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
hCPT
TPT
Typical experimental procedure for the synthesis of 2a–f
A solution of aldehyde (5 mmol), an appropriate urea
(7.5 mmol), 1,3-dione (7.5 mmol), and TMSCl (20 mmol) in dried
DMF (20 mL) was stirred at room temperature until TLC showed
complete disappearance of aldehyde. Reaction mixture was
poured into cold water (25 mL) and stirred for 10 min.
The precipitates formed were filtered and washed with cold
water (2 ꢂ 10 mL) and then with 90% ethanol (10 mL).
Recrystallization of the residues from an appropriate solvent
yielded target compounds 2 as white solids.
Typical experimental procedure for the synthesis of 3a–f
Compound 2 (0.5g) was dissolved in ethanol (15 mL), then dry
10% palladium on activated carbon (20 mg) was carefully added
to the solution and the dissolved oxygen was removed under
vacuum. Then a balloon of hydrogen was mounted and the
mixture was stirred vigorously for 12 h. The catalyst was
removed by filtration of the reaction mixture through a pad
of Celite. The filtrate was concentrated under reduced pressure
to give crude compounds 3 without further purification.
a
Cancer cells: A549: lung cancer cell; HCT116: colon cancer cell;
MDA-MB-435: breast cancer cell.
ꢅꢀꢀ
4b
4h
ꢄꢀ
4k
General experimental procedure for the synthesis of 4a–f
A
4m
ꢃꢀ
mixture of 3 (100 mg) and 7-formylhomocamptothecin
TPT
˚
(100 mg) was suspended in anhydrous CHCl3 containing 4 A
MS. After 10 min, Yb(OTf)3 (10 mg) was added to the solution.
The resulting mixture was stirred at room temperature until the
reaction was complete. After filtering the sieves, the solvent was
evaporated to dryness and purified by flash chromatography (eluent:
CH2Cl2/MeOH, 98:2) on silica gel to get target compound 4.
ꢂꢀ
ꢁꢀ
ꢀ
ꢀꢆꢀꢅ
ꢀꢆꢅ
ꢅ
ꢅꢀ
ꢅꢀꢀ
μg/ml
7-Formylhomocamptothecin
Figure 4. Comparison of the inhibition ratio of compounds 4b, 4h,
4k, 4m, and TPT against cancer cell line HCT116.
A yellow powder; mp > 3008C; IR (KBr) v: 3371, 3057, 2960, 2927,
1730, 1692, 1658, 1607, 1537, 1503, 1481, 1462, 1381, 1308,
1286, 1212, 1044, 839, 775. 1H-NMR (500 MHz, DMSO-d6) d:
0.87 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.87 (q, 2H, J ¼ 7.3 Hz,
CH3CH2), 3.04–3.49 (q, 2H, J ¼ 11.6 Hz, CH2CO), 5.43–5.55
(q, 2H, J ¼ 15.3 Hz, OCH2), 5.52 (s, 2H, NCH2), 6.06 (s, 1H, OH),
7.46 (s, 1H, C14-H), 7.92 (t, 1H, J ¼ 7.5 Hz, C10-H), 7.98 (t, 1H,
J ¼ 7.7 Hz, C11-H), 8.30 (d, 1H, J ¼ 8.5 Hz, C12-H), 9.04 (d, 1H,
J ¼ 8.6 Hz, C9-H). MS (ESI): 389 (MꢃH); Anal. calcd. for
C22H18N2O5: C, 67.69; H, 4.65; N, 7.18. Found: C, 67.57; H,
4.66; N, 7.15.
4a: A yellow powder; yield (77%); mp >3008C; IR (KBr) v: 3263,
3118, 2968, 2924, 2852, 1742, 1682, 1658, 1594, 1536, 1486,
1468, 1443, 1376, 1343, 1305, 1280, 1244, 1200, 1184, 842,
761, 721, 647. 1H-NMR (500 MHz, DMSO-d6) d: 0.89 (t, 3H,
J ¼ 7.3 Hz, CH3CH2), 0.92 (t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.87
Figure 5. Effect of the selected compounds on Topoisomerase-I
mediated DNA relaxation in single concentration. Topo I was
Topoisomerase I and DNA without drugs. The samples were
reacted with 100 mM drugs at 378C for 15 min. Reactions were then
stopped by adding 0.5% SDS. Gels were photographed under a
UV transilluminator.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Novel Homocamptothecins Conjugating with Dihydropyrimidine Derivatives
731
(q, 2H, J ¼ 8.4 Hz, CH3CH2), 3.04–3.49 (q, 2H, J ¼ 13.7 Hz,
CH2CO), 3.06 (s, 1H, COCH), 3.86 (q, 2H, J ¼ 6.9 Hz, CH3CH2O),
4.89 (d, 1H, J ¼ 11.4 Hz, NHCH), 5.43–5.59 (q, 2H, J ¼ 11.9 Hz,
OCH2), 5.59 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.30 (s, 1H, C14-H), 7.45
(s, 2H, NH), 7.50–7.60 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.69 (s, 1H, OH),
7.83 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.26
(d, 1H, J ¼ 8.4 Hz, C12-H), 8.99 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.72
(s, 1H, N ¼ CH). 13C-NMR (500 MHz, DMSO-d6) d: 8.65, 14.05,
29.43, 36.69, 42.80, 51.18, 53.03, 53.32, 60.82, 61.74, 73.56,
99.96, 122.29, 123.15, 124.56, 126.42, 128.86, 129.12, 129.55,
130.22, 130.86, 134.00, 138.32, 144.44, 148.05, 149.53, 153.58,
154.14, 156.18, 156.90, 159.42, 167.43, 172.27. MS (ESI):
718 (MꢃH); Anal. calcd. for C36H33F3N5O8: C, 60.08; H, 4.48;
N, 9.73. Found: C, 60.18; H, 4.47; N, 9.71.
4e: A yellow powder; yield (69%); mp >3008C; IR (KBr) v: 3311,
3080, 2978, 2923, 2851, 1739, 1656, 1612, 1537, 1500, 1480,
1439, 1376, 1338, 1282, 1249, 1186, 1057, 930, 906, 846, 767,
722. 1H NMR (500 MHz, DMSO-d6) d: 0.87 (t, 3H, J ¼ 7.3 Hz,
CH3CH2), 0.89 (t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.15 (t, 3H,
J ¼ 6.8 Hz, CH3CH2N), 1.87 (q, 2H, J ¼ 8.4 Hz, CH3CH2), 3.07–
3.49 (q, 2H, J ¼ 13.4 Hz, CH2CO), 3.06 (d, 1H, J ¼ 11.0 Hz,
COCH), 3.39–3.48 (d q, 2H, J ¼ 6.7 Hz, CH3CH2N), 3.83 (q, 2H,
J ¼ 7.1 Hz, CH3CH2O), 4.76 (d, 1H, J ¼ 11.0 Hz, NHCH), 5.40–5.56
(q, 2H, J ¼ 14.9 Hz, OCH2), 5.60 (s, 2H, NCH2), 6.04 (s, 1H, OH),
7.44 (s, 1H, C14-H), 7.46 (s, 1H, OH), 7.48–7.61 (dd, 4H, J ¼ 8.2 Hz,
Ar-H), 7.75 (s, 1H, NH), 7.85 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.94 (t, 1H,
J ¼ 7.9 Hz, C11-H), 8.27 (d, 1H, J ¼ 8.4 Hz, C12-H), 9.01 (d, 1H,
–
J ¼ 8.5 Hz, C -H), 9.72 (s, 1H, N CH). MS (ESI): 746 (MꢃH);
–
9
4b: A yellow powder; yield (90%); mp >3008C; IR (KBr) v: 3267,
3132, 2970, 2929, 1742, 1689, 1656, 1594, 1535, 1482, 1468,
1443, 1375, 1343, 1306, 1277, 1246, 1199, 1057, 1026, 851,
763, 698, 636. 1H-NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H,
J ¼ 7.3 Hz, CH3CH2), 0.91 (t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.88
(q, 2H, J ¼ 7.4 Hz, CH3CH2), 3.08–3.49 (q, 2H, J ¼ 13.7 Hz,
CH2CO), 3.10 (s, 1H, COCH), 3.87 (q, 2H, J ¼ 7.0 Hz, CH3CH2O),
4.94 (d, 1H, J ¼ 11.4 Hz, NHCH), 5.40–5.55 (q, 2H, J ¼ 14.95 Hz,
OCH2), 5.56 (s, 2H, NCH2), 6.06 (s, 1H, OH), 7.35 (1H, Ar-H), 7.37
(s, 1H, C14-H), 7.45 (s, 2H, NH), 7.48–7.59 (m, 3H, Ar-H), 7.73 (s, 1H,
OH), 7.84 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.93 (t, 1H, J ¼ 7.9 Hz, C11-H),
8.25 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.94 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.68
Anal. calcd. for C38H36F3N5O8: C, 61.04; H, 4.85; N, 9.37.
Found: C, 61.19; H, 4.86; N, 9.35.
4f: A yellow powder; yield (80%); mp >3008C; IR (KBr) v: 3310,
3151, 2972, 2930, 1742, 1701, 1654, 1594, 1535, 1508, 1441,
1370, 1311, 1277, 1227, 1085, 830, 796, 760, 721, 650. 1H-NMR
(500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.16
(t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.86 (q, 2H, J ¼ 7.9 Hz, CH3CH2),
2.28 (s, 3H, C ¼ C-CH3), 3.06–3.48 (q, 2H, J ¼ 13.8 Hz, CH2CO),
4.03 (q, 2H, J ¼ 7.0 Hz, CH3CH2O), 5.23 (d, 1H, J ¼ 3.1 Hz, NHCH),
5.40–5.56 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.57 (s, 2H, NCH2), 6.04 (s,
1H, OH), 7.38–7.54 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.45 (s, 1H, C14-H),
7.82 (s, 1H, NH), 7.84 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95 (t, 1H,
J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H,
J ¼ 8.5 Hz, C9-H), 9.25 (s, 1H, NH), 9.69 (s, 1H, N ¼ CH). 13C-
NMR (500 MHz, DMSO-d6) d: 8.65, 14.62, 18.28, 29.41, 36.69,
42.80, 53.01, 54.06, 59.75, 61.75, 73.56, 99.66, 99.96, 122.28,
123.14, 124.85, 125.69, 127.69, 128.91, 130.21, 131.05, 133.97,
144.44, 144.66, 149.00, 149.51, 150.78, 152.60, 153.55, 156.18,
156.97, 159.42, 165.80, 172.28. MS (ESI): 646 (MꢃH); Anal. calcd.
for C36H33N5O7: C, 66.76; H, 5.14; N, 10.81. Found: C, 66.63;
H, 5.15; N, 10.83.
4g: A yellow powder; yield (79%); mp >3008C; IR (KBr) v: 3239,
3111, 2970, 2933, 1737, 1702, 1654, 1616, 1594, 1535, 1506,
1481, 1442, 1369, 1311, 1281, 1223, 1087, 1055, 829, 761, 722,
634. 1H-NMR (500 MHz, DMSO-d6) d: 0.89 (t, 3H, J ¼ 7.3 Hz,
CH3CH2), 1.15 (t, 3H, J ¼ 7.0 Hz, CH3CH2O), 2.28 (q, 2H,
J ¼ 7.5 Hz, CH3CH2), 2.36 (s, 3H, C ¼ C-CH3), 3.04–3.48 (q, 2H,
J ¼ 13.7 Hz, CH2CO), 4.03 (q, 2H, J ¼ 6.9 Hz, CH3CH2O), 5.28
(s, 1H, NHCH), 5.53–5.58 (q, 2H, J ¼ 11.8 Hz, OCH2), 5.58 (s, 2H,
NCH2), 6.04 (s, 1H, OH), 7.35 (1H, Ar-H), 7.39 (1H, Ar-H), 7.46 (s, 1H,
C14-H), 7.48–7.50 (2H, Ar-H), 7.84 (s, 1H, NH), 7.85 (t, 1H,
J ¼ 7.9 Hz, C10-H), 8.03 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.27 (d, 1H,
J ¼ 8.3 Hz, C12-H), 8.96 (d, 1H, J ¼ 8.4 Hz, C9-H), 9.25 (s, 1H,
NH), 9.67 (s, 1H, N ¼ CH). 13C-NMR (500 MHz, DMSO-d6)
d: 8.65, 14.62, 18.28, 36.69, 42.80, 53.00, 54.31, 59.75, 61.72,
73.55, 99.46, 99.95, 119.58, 121.68, 123.14, 124.89, 125.01,
125.57, 128.90, 129.15, 130.09, 130.26. 130.89, 133.14, 144.44,
146.60, 149.14, 149.87, 151.23, 152.54, 153.62, 156.42, 157.26,
159.42, 165.79, 172.25. MS (ESI): 646 (MꢃH); Anal. calcd. for
C36H33N5O7: C, 66.76; H, 5.14; N, 10.81. Found: C, 66.65;
H, 5.16; N, 10.85.
(s, 1H, N CH). 13C-NMR (500 MHz, DMSO-d6) d: 8.65, 14.04, 29.43,
–
–
36.69, 40.91, 43.22, 51.11, 53.03, 53.59, 60.75, 61.75, 73.56, 99.99,
121.98, 122.24, 123.17, 124.58, 125.89, 127.12, 128.90, 129.13,
129.83, 130.04, 130.87, 133.95, 140.26, 144.41, 149.53, 151.11,
153.58, 154.15, 156.19, 157.02, 159.42, 167.49, 172.27. MS (ESI):
718 (MꢃH); Anal. calcd. for C36H33F3N5O8: C, 60.08; H, 4.48;
N, 9.73. Found: C, 60.22; H, 4.47; N, 9.71.
4c: A yellow powder; yield (83%); mp >3008C; IR (KBr) v: 3252,
3093, 2978, 2933, 1742, 1660, 1596, 1537, 1470, 1376, 1341,
1301, 1274, 1237, 1188, 1140, 1058, 847, 766. 1H-NMR
(500 MHz, DMSO-d6) d: 0.89 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 0.88
(t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.87 (q, 2H, J ¼ 8.4 Hz, CH3CH2),
2.9 (s, 3H, NCH3), 3.07–3.49 (q, 2H, J ¼ 13.8 Hz, CH2CO), 3.22 (d,
1H, J ¼ 11.2 Hz, COCH), 3.84 (q, 2H, J ¼ 6.9 Hz, CH3CH2O), 4.77
(d, 1H, J ¼ 11.1 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 11.8 Hz, OCH2),
5.60 (s, 2H, NCH2), 6.05 (s, 1H, OH), 7.46 (s, 1H, C14-H), 7.49 (s, 1H,
OH), 7.50–7.61 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.81 (s, 1H, NH), 7.84
(t, 1H, J ¼ 7.9 Hz, C10-H), 7.96 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.27
(d, 1H, J ¼ 8.4 Hz, C12-H), 9.01 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.72 (s, 1H,
N ¼ CH). MS (ESI): 732 (MꢃH); Anal. calcd. for C37H34F3N5O8: C, 60.57;
H, 4.67; N, 9.55. Found: C, 60.67; H, 4.68; N, 9.57.
4d: A yellow powder; yield (77%); mp >3008C; IR (KBr) v: 3338,
3059, 2966, 2923, 1744, 1655, 1597, 1498, 1440, 1371, 1312,
1277, 1223, 1208, 1053, 831, 759, 693, 632. 1H-NMR (500 MHz,
DMSO-d6) d: 0.89 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 0.92 (t, 3H,
J ¼ 7.0 Hz, CH3CH2O), 1.87 (q, 2H, J ¼ 8.4 Hz, CH3CH2), 3.07–
3.48 (q, 2H, J ¼ 13.7 Hz, CH2CO), 3.86 (q, 2H, J ¼ 6.9 Hz,
CH3CH2O), 4.38 (s, 1H, CHCO), 4.39 (s, 1H, NHCH), 5.40–5.56
(q, 2H, J ¼ 11.9 Hz, OCH2), 5.60 (s, 2H, NCH2), 6.04 (s, 1H, OH),
6.68 (t, 1H, Ar-H), 6.90 (t, 1H, Ar-H), 7.23 (t, 2H, Ar-H), 7.41 (d, 1H,
Ar-H), 7.43 (s, 1H, OH), 7.45 (s, 1H, C14-H), 7.46–7.58 (dd, 4H,
J ¼ 8.3 Hz, Ar-H), 7.84 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95 (t, 1H,
J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.60 (s, 1H,
4h: A yellow powder; yield (87%); mp >3008C; IR (KBr) v: 3315,
2926, 1743, 1699, 1658, 1594, 1537, 1515, 1435, 1371, 1345,
1278, 1237, 1078, 1053, 972, 829, 760, 722. 1H-NMR (500 MHz,
DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.86 (q, 2H,
–
–
NH), 9.00 (d, 1H, J ¼ 8.5 Hz, C -H), 9.72 (s, 1H, N CH). MS (ESI):
J ¼ 7.7 Hz, CH CH ), 2.29 (s, 3H, C C–CH ), 3.07–3.48 (q, 2H,
–
–
9
3
2
3
794 (MꢃH); Anal. calcd. for C42H36F3N5O8: C, 63.39; H, 4.56;
J ¼ 13.9 Hz, CH2CO), 3.57 (s, 3H, CH3O), 5.23 (d, 1H,
J ¼ 3.4 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 14.9 Hz, OCH2), 5.57
N, 8.80. Found: C, 63.48; H, 4.55; N, 8.78.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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732
L.-J. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
(s, 2H, NCH2), 6.04 (s, 1H, OH), 7.38–7.54 (dd, 4H, J ¼ 8.4 Hz, Ar-H),
7.45 (s, 1H, C14-H), 7.81 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.82 (s, 1H, NH),
7.95 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H, J ¼ 7.7 Hz, C12-H), 8.98
H), 7.95 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.24 (d, 1H, J ¼ 3.5 Hz, NH), 8.26
(d, 1H, J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.72
(s, 1H, N ¼ CH). MS (ESI): 722 (MꢃH); Anal. calcd. for C42H37N5O7:
C, 69.70; H, 5.15; N, 9.68. Found: C, 69.84; H, 5.14; N, 9.66.
–
(d, 1H, J ¼ 8.5 Hz, C -H), 9.27 (s, 1H, NH), 9.68 (s, 1H, N CH). MS
–
9
(ESI): 632 (MꢃH); Anal. calcd. for C35H31N5O7: C, 66.34; H, 4.93; N,
4m: A yellow powder; yield (88%); mp >3008C; IR (KBr) v: 3352,
3253, 3132, 2973, 2931, 1746, 1698, 1653, 1595, 1533, 1510,
1444, 1368, 1343, 1315, 1278, 1235, 1169, 1090, 1057, 850,
760. 1H-NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz,
CH3CH2), 1.36 (s, 9H, OC(CH3)3), 1.87 (q, 2H, J ¼ 7.9 Hz,
11.05. Found: C, 66.47; H, 4.92; N, 11.07.
4i: A yellow powder; yield (78%); mp >3008C; IR (KBr) v: 3276,
2926, 1743, 1702, 1655, 1597, 1535, 1509, 1439, 1381, 1366,
1329, 1315, 1278, 1236, 1174, 1136, 1110, 1055, 849, 830, 763,
1
–
722, 579. H-NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz,
CH CH ), 2.25 (s, 3H, C C–CH ), 3.07–3.49 (q, 2H, J ¼ 13.9 Hz,
–
3
2
3
CH3CH2), 1.87 (q, 2H, J ¼ 7.5 Hz, CH3CH2), 2.16 (s, 3H, OCCH3),
2.24 (s, 3H, C ¼ C-CH3), 3.06–3.48 (q, 2H, J ¼ 13.6 Hz, CH2CO),
5.35 (d, 1H, J ¼ 3.2 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 15.0 Hz,
OCH2), 5.57 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.39–7.53 (dd, 4H,
J ¼ 8.3 Hz, Ar-H), 7.45 (s, 1H, C14-H), 7.82 (t, 1H, J ¼ 8.0 Hz, C10-H),
7.91 (s, 1H, NH), 7.95 (t, 1H, J ¼ 8.1 Hz, C11-H), 8.26 (d, 1H,
J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.24 (s, 1H,
NH), 9.68 (s, 1H, N ¼ CH). MS (ESI): 616 (MꢃH); Anal. calcd. for
C35H31N5O6: C, 68.06; H, 5.06; N, 11.34. Found: C, 68.17; H, 5.05;
N, 11.32.
CH2CO), 4.03 (q, 2H, J ¼ 7.0 Hz, CH3CH2O), 5.18 (d, 1H,
J ¼ 3.4 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 15.0 Hz, OCH2), 5.58
(s, 2H, NCH2), 6.03 (s, 1H, OH), 7.38–7.56 (dd, 4H, J ¼ 8.3 Hz,
Ar-H), 7.45 (s, 1H, C14-H), 7.74 (s, 1H, NH), 7.83 (t, 1H,
J ¼ 7.9 Hz, C10-H), 7.94 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H,
J ¼ 8.4 Hz, C12-H), 8.99 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.12 (s, 1H,
–
–
NH), 9.70 (s, 1H, N CH). MS (ESI): 674 (MꢃH); Anal. calcd. for
C38H37N5O7: C, 67.54; H, 5.52; N, 10.36. Found: C, 67.68; H, 5.51;
N, 10.33.
4n: A yellow powder; yield (87%); mp >3008C; IR (KBr) v: 3310,
2960, 2931, 2873, 1742, 1705, 1682, 1651, 1594, 1537, 1510,
1440, 1396, 1377, 1312, 1276, 1234, 1081, 976, 829, 799, 760,
721, 631. 1H-NMR (500 MHz, DMSO-d6) d: 0.81–0.83 (d d, 6H,
J ¼ 5.3 Hz, CH(CH3)2), 0.88 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.16
(t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.85 (m, 1H, CH(CH3)2), 1.86
4j: A yellow powder; yield (76%); mp >3008C; IR (KBr) v: 3346,
3095, 2979, 2932, 1751, 1680, 1659, 1608, 1532, 1509, 1464,
1441, 1378, 1314, 1277, 1229, 1171, 1082, 1054, 854, 759. 1H-
NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.12
(t, 3H, J ¼ 6.9 Hz, CH3CH2N), 1.17 (t, 3H, J ¼ 7.0 Hz, CH3CH2O),
–
–
1.87 (q, 2H, J ¼ 7.8 Hz, CH CH ), 2.54 (s, 3H, C C–CH ), 3.07–3.48
(q, 2H, J ¼ 7.9 Hz, CH CH ), 2.31 (s, 3H, C C–CH ), 3.07–3.45
–
–
3
2
3
3
2
3
(q, 2H, J ¼ 13.9 Hz, CH2CO), 4.01–4.05 (d q, 2H, J ¼ 7.0 Hz,
CH3CH2N), 4.06 (q, 2H, J ¼ 7.0 Hz, CH3CH2O), 5.22 (d, 1H,
J ¼ 3.5 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.57
(s, 2H, NCH2), 6.03 (s, 1H, OH), 7.36–7.54 (dd, 4H, J ¼ 8.3 Hz,
Ar-H), 7.45 (s, 1H, C14-H), 7.82 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95
(t, 1H, J ¼ 7.9 Hz, C11-H), 7.97 (s, 1H, NH), 8.26 (d, 1H,
J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.69 (s, 1H,
(q, 2H, J ¼ 13.8 Hz, CH2CO), 3.78 (d, 1H, J ¼ 6.0 Hz, OCH), 5.24
(d, 1H, J ¼ 3.0 Hz, NHCH), 5.40–5.57 (q, 2H, J ¼ 15.4 Hz, OCH2),
5.58 (s, 2H, NCH2), 6.03 (s, 1H, OH), 7.38–7.54 (dd, 4H, J ¼ 8.3 Hz,
Ar-H), 7.45 (s, 1H, C14-H), 7.81 (s, 1H, NH), 7.84 (t, 1H,
J ¼ 7.9 Hz, C10-H), 7.95 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H,
J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.26 (s, 1H,
–
–
NH), 9.68 (s, 1H, N CH). MS (ESI): 674 (MꢃH); Anal. calcd. for
–
–
N CH). MS (ESI): 674 (MꢃH); Anal. calcd. for C38H37N5O7: C,
C38H37N5O7: C, 67.54; H, 5.52; N, 10.36. Found: C, 67.69; H, 5.51;
N, 10.38.
67.54; H, 5.52; N, 10.36. Found: C, 67.65; H, 5.53; N, 10.33.
4k: A yellow powder; yield (67%); mp >3008C; IR (KBr) v: 3307,
2960, 2925, 2852, 1746, 1674, 1659, 1593, 1534, 1507, 1458,
1443, 1383, 1349, 1302, 1269, 1246, 1173, 1075, 1053, 972,
857, 829, 806, 759, 821, 629. 1H-NMR (500 MHz, DMSO-d6)
d: 0.88 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.18 (t, 3H, J ¼ 7.0 Hz,
CH3CH2O), 1.87 (q, 2H, J ¼ 7.8 Hz, CH3CH2), 2.52 (s, 3H,
4o: A yellow powder; yield (85%); mp >3008C; IR (KBr) v: 3400,
3230, 3116, 2963, 2933, 2879, 1745, 1717, 1682, 1655, 1594,
1535, 1508, 1460, 1432, 1391, 1375, 1282, 1234, 1078, 1053,
997, 853, 800, 761, 719, 631. 1H-NMR (500 MHz, DMSO-d6)
d: 0.89 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.12 (t, 3H, J ¼ 7.0 Hz,
CH3CH2N), 1.87 (q, 2H, J ¼ 7.6 Hz, CH3CH2), 2.54 (s, 3H,
–
–
–
C C–CH ), 3.06–3.48 (q, 2H, J ¼ 13.8 Hz, CH CO), 3.28 (s, 3H,
C C–CH ), 3.07–3.48 (q, 2H, J ¼ 13.9 Hz, CH CO), 3.61 (s, 3H,
–
3
2
3
2
NCH3), 4.07 (q, 2H, J ¼ 7.0 Hz, CH3CH2O), 5.24 (d, 1H,
J ¼ 3.8 Hz, NHCH), 5.40–5.56 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.57
(s, 2H, NCH2), 6.03 (s, 1H, OH), 7.36–7.53 (dd, 4H, J ¼ 8.3 Hz,
Ar-H), 7.45 (s, 1H, C14-H), 7.82 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95
(t, 1H, J ¼ 7.9 Hz, C11-H), 8.04 (d, 1H, J ¼ 3.8 Hz, NH), 8.26
(d, 1H, J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H),
9.69 (s, 1H, N ¼ CH). MS (ESI): 660 (MꢃH); Anal. calcd. for
C37H35N5O7: C, 67.16; H, 5.33; N, 10.58. Found: C, 67.26; H,
5.32; N, 10.55.
OCH3), 3.63–3.85 (d q, 2H, J ¼ 7.0 Hz, CH3CH2N), 5.22 (d, 1H,
J ¼ 3.5 Hz, NHCH), 5.39–5.55 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.54
(s, 2H, NCH2), 6.03 (s, 1H, OH), 7.36–7.53 (dd, 4H, J ¼ 8.3 Hz,
Ar-H), 7.44 (s, 1H, C14-H), 7.81 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.93
(t, 1H, J ¼ 7.9 Hz, C11-H), 7.98 (d, 1H, J ¼ 3.7 Hz, NHCH), 8.23
(d, 1H, J ¼ 8.4 Hz, C12-H), 8.95 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.66
(s, 1H, N CH). 13C-NMR (500 MHz, DMSO-d6) d: 8.54, 15.21,
–
–
15.98, 37.35, 38.15, 42.78, 51.49, 52.54, 52.87, 61.68, 73.48,
99.88, 102.96, 122.26, 123.08, 125.78, 126.14, 127.43, 128.75,
128.95, 130.13, 130.75, 133.97, 143.75, 144.36, 149.61, 150.22,
152.89, 153.50, 156.11, 156.93, 159.36, 166.48, 172.13. MS (ESI):
660 (MꢃH); Anal. calcd. for C37H35N5O7: C, 67.16; H, 5.33;
N, 10.58. Found: C, 67.26; H, 5.32; N, 10.60.
4l: A yellow powder; yield (70%); mp >3008C; IR (KBr) v: 3344,
3095, 3066, 2974, 2934, 1745, 1707, 1686, 1655, 1618, 1593,
1535, 1497, 1438, 1372, 1343, 1311, 1268, 1210, 1079, 973,
858, 829, 761, 699, 632. 1H-NMR (500 MHz, DMSO-d6) d: 0.89 (t,
3H, J ¼ 7.3 Hz, CH3CH2), 1.19 (t, 3H, J ¼ 7.0 Hz, CH3CH2O), 1.88
4p: A yellow powder; yield (77%); mp >3008C; IR (KBr) v: 3351,
1968, 2929, 2883, 1744, 1687, 1655, 1593, 1535, 1508, 1439,
1421, 1381, 1348, 1270, 1243, 1206, 1072, 1056, 933, 852, 830,
–
(q, 2H, J ¼ 7.8 Hz, CH CH ), 2.07 (s, 3H, C C–CH ), 3.07–3.46 (q,
–
3
2
3
2H, J ¼ 13.8 Hz, CH2CO), 3.28 (s, 3H, NCH3), 4.09 (q, 2H,
J ¼ 7.0 Hz, CH3CH2O), 5.37 (d, 1H, J ¼ 3.5 Hz, NHCH), 5.40–
5.56 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.58 (s, 2H, NCH2), 6.05 (s, 1H,
OH), 7.26 (d, 2H, Ar-H), 7.41–7.54 (m, 3H, Ar-H), 7.46 (s, 1H, C14-H),
1
760, 721. H-NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz,
CH3CH2), 1.87 (q, 2H, J ¼ 7.5 Hz, CH3CH2), 2.17 (s, 3H, OCCH3),
–
2.50 (s, 3H, C C–CH ), 3.06–3.48 (q, 2H, J ¼ 13.6 Hz, CH CO), 3.12
–
3
2
7.55–7.63 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.82 (t, 1H, J ¼ 7.9 Hz, C10
-
(s, 3H, NCH3), 5.30 (d, 1H, J ¼ 3.7 Hz, NHCH), 5.39–5.52 (q, 2H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
Novel Homocamptothecins Conjugating with Dihydropyrimidine Derivatives
733
J ¼ 15.0 Hz, OCH2), 5.54 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.39–7.52
(dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.45 (s, 1H, C14-H), 7.81 (t, 1H,
J ¼ 8.0 Hz, C10-H), 7.94 (t, 1H, J ¼ 8.1 Hz, C11-H), 8.13 (d, 1H,
J ¼ 3.9 Hz, NH), 8.25 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.96 (d, 1H,
J ¼ 8.0 Hz, C10-H), 7.94 (t, 1H, J ¼ 8.1 Hz, C11-H), 8.05 (d, 1H,
J ¼ 3.3 Hz, NHCH), 8.26 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H,
J ¼ 8.5 Hz, C9-H), 9.69 (s, 1H, N ¼ CH). MS (ESI): 644 (MꢃH); Anal.
calcd. for C37H35N5O6: C, 68.82; H, 5.46; N, 10.85. Found: C, 68.97;
H, 5.45; N, 10.83.
–
J ¼ 8.5 Hz, C -H), 9.67 (s, 1H, N CH). MS (ESI): 630 (MꢃH);
–
9
Anal. calcd. for C36H33N5O6: C, 68.45; H, 5.27; N, 11.09. Found:
C, 68.59; H, 5.28; N, 11.11.
Cytotoxicity
4q: A yellow powder; yield (88%); mp >3008C; IR (KBr) v: 3334,
2972, 2930, 2883, 1746, 1674, 1597, 1536, 1510, 1435, 1389,
1369, 1344, 1283, 1232, 1140, 1080, 1053, 950, 851, 759,
821, 630. 1H-NMR (500 MHz, DMSO-d6) d: 0.89 (t, 3H,
J ¼ 7.3 Hz, CH3CH2), 1.11 (t, 3H, J ¼ 6.9 Hz, CH3CH2N), 1.37
(s, 9H, C(CH3)3), 1.87 (q, 2H, J ¼ 7.8 Hz, CH3CH2), 2.54 (s, 3H,
One thousand two hundred cells per well were plated in 96-well
plates. After culturing for 24 h, test compounds were added onto
triplicate wells with different concentration, and 0.1% DMSO for
control. After three days of incubation, 20 mL MTT (3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) solution
(5 mg/mL) was added to each well, and after shaking for
1 min the plate was incubated further for 4 h. Formazan crystals
were dissolved with 100 mL DMSO. The absorbance (OD) was
quantitated with microplate spectrophotometer at 570 nm.
Wells containing no drugs were used as blanks for the spectro-
photometer. The survival of the cells was expressed as percentage
of untreated control wells.
–
C C–CH ), 3.07–3.48 (q, 2H, J ¼ 13.9 Hz, CH CO), 3.60–3.86
–
3
2
(d q, 2H, J ¼ 7.0 Hz, CH3CH2N), 5.17 (d, 1H, J ¼ 3.3 Hz, NHCH),
5.40–5.56 (q, 2H, J ¼ 14.9 Hz, OCH2), 5.58 (s, 2H, NCH2), 6.03
(s, 1H, OH), 7.36–7.57 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.45 (s, 1H,
C14-H), 7.82 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.88 (d, 1H, J ¼ 3.6 Hz,
NHCH), 7.94 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.26 (d, 1H,
J ¼ 8.4 Hz, C12-H), 8.99 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.70 (s, 1H,
–
N CH). MS (ESI): 702 (MꢃH); Anal. calcd. for C40H41N5O7: C,
–
68.26; H, 5.87; N, 9.95. Found: C, 68.39; H, 5.88; N, 9.92.
Topoisomerase I inhibition activity assays
4r: A yellow powder; yield (78%); mp >3008C; IR (KBr) v: 3339,
2961, 2929, 2873, 1746, 1677, 1659, 1597, 1535, 1511, 1462,
1443, 1380, 1270, 1246, 1073, 1051, 829, 760, 721, 629.
1H-NMR (500 MHz, DMSO-d6) d: 0.83 (d d, 6H, J ¼ 6.5 Hz,
CH(CH3)2), 0.88 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.83 (m, 1H,
CH(CH3)2), 1.87 (q, 2H, J ¼ 7.8 Hz, CH3CH2), 2.50 (s, 3H,
Camptothecin was obtained from the company of
Tianzunzezhong in China. Topo I (calf thymus), buffer, BSA,
loading buffer and supercoiled DNA pBR322 were all from
TaKaRa Biotechnology CO., Ltd.
All reactions were carried out in 20-mL volumes (16 mL double
distilled water, 2 mL DNA Topo I buffer, 2 mL 0.1% BSA) including
0.25 mg supercoiled DNA, 0.5 U Topo I with or without drug. The
reaction was incubated at 378C for 15 min. Reactions were
stopped by adding SDS (0.5% final concentration). To the reaction
mixtures, 3.5 mL 6 ꢂ loading buffer (0.1 mM EDTA, 7% glycerol,
0.01% xylene cyanol FF, Bromophenol Blue 0.01%) was added. The
mixtures were electrophoresed in 0.8% agarose gel in TAE
buffer for 40 min at 120 V. The gel was stained with ethidium
bromide at room temperature and photographed with UV
transilluminator.
–
C C–CH ), 3.08–3.48 (q, 2H, J ¼ 13.8 Hz, CH CO), 3.14 (s, 3H,
–
3
2
NCH3), 3.83 (d, 2H, J ¼ 6.3 Hz, OCH2), 5.26 (d, 1H, J ¼ 3.7 Hz,
NHCH), 5.39–5.55 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.46 (d, 2H,
J ¼ 3.9 Hz, NCH2), 6.05 (s, 1H, OH), 7.37–7.52 (dd, 4H,
J ¼ 8.3 Hz, Ar-H), 7.42 (s, 1H, C14-H), 7.71 (t, 1H,
J ¼ 7.9 Hz, C10-H), 7.90 (t, 1H, J ¼ 7.9 Hz, C11-H), 8.05 (d, 1H,
J ¼ 3.8 Hz, NH), 8.20 (d, 1H, J ¼ 8.4 Hz, C12-H), 8.89 (d, 1H,
–
J ¼ 8.5 Hz, C -H), 9.61 (s, 1H, N CH). MS (ESI): 660 (MꢃH);
–
9
Anal. calcd. for C39H39N5O7: C, 67.91; H, 5.70; N, 10.15. Found:
C, 67.85; H, 5.69; N, 10.13.
4s: A yellow powder; yield (78%); mp >3008C; IR (KBr) v: 3314,
3219, 2974, 2921, 2850, 1740, 1701, 1655, 1692, 1551, 1498,
1448, 1355, 1158, 854, 752, 697, 621. 1H-NMR (500 MHz,
DMSO-d6) d: 0.89 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.39 (s, 9H,
OC(CH3)3), 1.88 (q, 2H, J ¼ 7.8 Hz, CH3CH2), 2.02 (s, 3H,
This research was supported in part by the National Natural Science
Foundation of China (No. 30371689), the Key Project of Science and
Technology of Shanghai (No. 09431901700), Major Special Project for
the Creation of New Drugs (Grant Nos. 2009ZX09301-011).
–
C C–CH ), 3.07–3.49 (q, 2H, J ¼ 13.8 Hz, CH CO), 5.31 (d, 1H,
–
3
2
The authors have declared no conflict of interest.
J ¼ 3.2 Hz, NHCH), 5.44–5.57 (q, 2H, J ¼ 15.4 Hz, OCH2), 5.61
(s, 2H, NCH2), 6.04 (s, 1H, OH), 6.87 (t, 2H, Ar-H), 7.41–7.54
(t, 2H, Ar-H), 7.38 (d, 1H, Ar-H), 7.45 (s, 1H, C14-H), 7.53–7.65
(dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.82 (t, 1H, J ¼ 7.9 Hz, C10-H), 7.95
(t, 1H, J ¼ 7.9 Hz, C11-H), 8.16 (d, 1H, J ¼ 3.5 Hz, NH), 8.27
(d, 1H, J ¼ 8.4 Hz, C12-H), 8.98 (d, 1H, J ¼ 8.5 Hz, C9-H), 9.75
References
[1] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T.
McPhail, G. A. Sim, J. Am. Chem. Soc. 1966, 88, 3888–3890.
–
(s, 1H, N CH). MS (ESI): 750 (MꢃH); Anal. calcd. for C44H41N5O7:
–
C, 70.29; H, 5.50; N, 9.32. Found: C, 70.43; H, 5.51; N, 9.35.
4t: A yellow powder; yield (65%); mp >3008C; IR (KBr) v: 3348,
3061, 2971, 2924, 2851, 1744, 1676, 1655, 1591, 1535, 1509,
1387, 1373, 1279, 1226, 1170, 1030, 850, 831, 760, 721, 638.
1H-NMR (500 MHz, DMSO-d6) d: 0.88 (t, 3H, J ¼ 7.4 Hz, CH3CH2),
1.11 (t, 3H, J ¼ 6.7 Hz, NCH2CH3), 1.87 (q, 2H, J ¼ 7.5 Hz,
[2] Y. H. Hsiang, R. Hertzberg, S. Hecht, L. F. Liu, J. Biol. Chem.
1985, 260, 14873–14878.
[3] Y. H. Hsiang, M. G. Lihou, L. F. Liu, Cancer Res. 1989, 49, 5077–
5082.
[4] H. Ulukan, P. W. Swaan, Drugs 2002, 62, 2039–2057.
[5] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, Bioorg. Med. Chem.
Lett. 1997, 7, 2235–2238.
–
CH CH ), 2.16 (s, 3H, OCCH ), 2.24 (s, 3H, C C–CH ), 3.06–3.48
–
3
2
3
3
(q, 2H, J ¼ 13.6 Hz, CH2CO), 3.62–3.85 (d q, 2H, J ¼ 7.0 Hz,
NCH2CH3), 5.28 (d, 1H, J ¼ 3.5 Hz, NHCH), 5.40–5.56 (q, 2H,
J ¼ 15.0 Hz, OCH2), 5.56 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.39–
7.55 (dd, 4H, J ¼ 8.3 Hz, Ar-H), 7.45 (s, 1H, C14-H), 7.82 (t, 1H,
[6] C. Bailly, A. Lansiaux, L. Dassonneville, D. Demarquay,
H. Coulomb, M. Huchet, O. Lavergne, D. C. Bigg,
Biochemistry 1999, 38, 15556–15563.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

734
L.-J. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 726–734
[7] O. Lavergne, L. Lesueur-Ginot, R. F. Pla, P. G. Kasprzyk, J.
Pommier, D. Demarquay, G. Prevost, G. Ulibarri, A. Rolland,
A. M. Schiano-Liberatore, J. Harnett, D. Pons, J. Camara, D. C.
Bigg, J. Med. Chem. 1998, 41, 5410–5419.
M. Gigli, R. Angelucci, A. Suarato, J. Controlled Release 2000,
65, 105–119.
[17] M. A. Walker, G. M. Dubowchik, S. J. Hofstead, P. A. Trail,
R. A. Firestone, Bioorg. Med. Chem. Lett. 2002, 12, 217–219.
[8] O. Lavergne, D. Demarquay, C. Bailly, C. Lanco, A. Rolland,
M. Huchet, H. Coulomb, N. Muller, N. Baroqqi, J. Camara, C.
Le Breton, E. Manqinot, J. B. Cazaux, D. C. Bigg, J. Med. Chem.
2000, 43, 2285–2289.
[18] J.-Y. Chang, X. Guo, H.-X. Chen, Z. Jiang, Q. Fu, H.-K. Wang,
K. F. Bastow, X.-K. Zhu, J. Guan, K.-H. Lee, Y.-C. Cheng,
Biochem. Pharmacol. 2000, 59, 497–508.
[19] C. O. Kappe, Acc. Chem. Res. 2000, 33, 879–888; and references
cited therein.
[9] R. S. Tangirala, S. Antony, K. Agama, Y. Pommier, B. D.
Anderson, R. Bevins, D. P. Curran, Bioorg. Med. Chem. 2006,
14, 6202–6212.
[20] B. Jauk, T. Pernat, C. O. Kappe, Molecules 2000, 5, 227–239.
[21] A. D. Patil, N. V. Kumar, W. C. Kokke, M. F. Bean, A. J. Freyer,
C. Debrossi, S. Mai, A. Truneh, D. J. Faulkner, B. Carte, A. L.
Breen, R. P. Hertzberg, R. K. Johnson, J. W. Westley, B. C. M.
Potts, J. Org. Chem. 1995, 60, 1182–1188.
[10] H. Gelderblom, R. Salazar, J. Verweij, G. Pentheroudakis,
M. J. de Jonge, M. Devlin, C. van Hooije, F. Seguy, R. Obach, J.
Prunonosa, P. Principe, C. Twelves, Clin. Cancer Res. 2003, 9,
4101–4107.
[22] B. B. Snider, J. Chen, A. D. Patil, A. Freyer, Tetrahedron Lett.
1996, 37, 6977–6980.
[11] D. Demarquay, M. Huchet, H. Coulomb, L. Lesueur-Ginot, O.
Lavergne, J. Camara, P. G. Kasprzyk, G. Prevost, D. C. Bigg,
Cancer Res. 2004, 64, 4942–4949.
[23] T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King, S. I.
Schreiber, T. J. Mitchison, Science 1999, 268, 971–974.
[12] L. J. Zhu, Z. Y. Miao, C. Q. Sheng, W. Guo, J. Z. Yao, W. F. Liu,
X. Y. Che, W. Y. Wang, P. F. Cheng, W. N. Zhang, Eur. J. Med.
Chem. 2010, 45, 2726–2732.
[24] D. Russowsky, R. F. S. Canto, S. A. A. Sanches, M. G. M. DOca,
A. de Fatima, R. A. Pilli, L. K. Kohn, M. A. Antonio, J. E.
de Carvalho, Bioorg. Chem. 2006, 34, 173–182.
[13] Z. Y. Miao, C. Q. Sheng, W. N. Zhang, H. T. Ji, J. Zhang, L. C.
Shao, L. You, M. Zhang, J. Z. Yao, X. Y. Che, Bioorg. Med. Chem.
2008, 16, 1493–1510.
[25] P. Hamet, Hypertension 1996, 28, 321–324.
[26] E. Grossman, F. H. Messerli, V. Boyko, U. Goldbourt, Am. J.
Med. 2002, 112, 479–486.
[14] Z. Y. Miao, J. Zhang, L. You, J. Wang, C. Q. Sheng, J. Z.
Yao, W. N. Zhang, H. Feng, W. Guo, L. Zhou, W. F. Liu,
L. J. Zhu, P. F. Cheng, X. Y. Che, W. Y. Wang, C. Luo,
Y. L. Xu, G. Q. Dong, Bioorg. Med. Chem. 2010, 18, 3140–
3146.
[27] B. L. Staker, M. D. Feese, M. Cushman, Y. Pommier, D.
Zembower, L. Stewart, A. B. Burgin, J. Med. Chem. 2005, 48,
2336–2345.
[28] S. V. Ryabukhin, A. S. Plaskon, E. N. Ostapchuk, D. M.
Volochnyuk, O. V. Shishkin, A. A. Tolmachev, J. Fluorine
Chem. 2008, 129, 625–631.
[15] F. M. H. de Groot, G. F. Busscher, R. W. M. Aben, H. W.
Scheeren, Bioorg. Med. Chem. Lett. 2002, 12, 2371–2376.
[29] S. Sawada, K. Nokata, T. Furuta, T. Yokokura, T. Miyasaka,
Chem. Pharm. Bull. 1991, 39, 2574–2580.
[16] V. R. Caiolfa, M. Zamai, A. Fiorino, E. Frigerio, C. Pellizzoni,
R. d’Argy, A. Ghiglieri, M. G. Castelli, M. Farao, E. Pesenti,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com