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Synthesis Telomerase Evaluation and Anti-Proliferative Studies on Various Series of Diaminoanthraquinone-Linked Aminoacyl Residue Derivatives.

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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
101
Full Paper
Synthesis, Telomerase Evaluation and Anti-Proliferative
Studies on Various Series of Diaminoanthraquinone-Linked
Aminoacyl Residue Derivatives
Fong-Chun Huang1, Kuo-Feng Huang2, Ruey-Hui Chen3, Jia-Er Wu4, Tsung-Chih Chen5,
Chun-Liang Chen5, Chia-Chung Lee5, Jin-Yang Chen5, Jing-Jer Lin1, and Hsu-Shan Huang4,5
1
Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan
Chi-Mei Medical Center, Tainan, Taiwan
3
College of Nursery, Hungkuang University, Taichung, Taiwan
4
School of Pharmacy, Taipei, Taiwan
5
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
2
Four series of compounds containing an anthraquinone-linked moiety and symmetrical or
asymmetrical aminoacyl residues in side chains at positions 1,4-, 1,5-, 2,6-, and 2,7- were
synthesized and evaluated for their inhibitory effects toward telomerase and hTERT expression. Of
these, only compound B11 showed selective inhibition of telomerase activity. Although it is not as
competent as several of the anthraquinones we identified previously, nevertheless, the result is
consistent with that the general structure moiety at the 1,5-position of diaminoanthraquinonelinked compound is important for the telomerase inhibitory activity. Interestingly, compounds
A6, A8, C8, and D8 exhibited selective repressing activities toward hTERT expression and showed
less effect toward proliferation of the treated cancer cells. Although it is not apparent which structure
moiety is responsible for the telomerase repression effects of these compounds, they could be further
developed as potential anti-tumor agents.
Keywords: Anthraquinone-linkage / Dose-dependent pattern / SEAP expression / Telomerase / TRAP assay
Received: April 5, 2011; Revised: May 3, 2011; Accepted: May 3, 2011
DOI 10.1002/ardp.201100122
Introduction
G-Quadruplex structures are formed by associating four guanine bases through a stable hydrogen bond arrangement.
They are formed in DNA or RNA rich in guanine-residues [1].
The vertebrate telomeric DNA sequences are short tandem
repeats of TTAGGG sequences. They are capable of forming
G-quadruplex structures. Telomerase is an enzyme which
synthesizes the G-rich strand of telomere DNA. The presence
of telomerase activity is highly correlated with cancer formation and is considered as a means that allows cancer cells
to escape senescence [2]. It is elevated in more than 85% of
cancer cells, thus providing a compelling rationale to target
Correspondence: Prof. Dr. Hsu-Shan Huang, School of Pharmacy,
National Defense Medical Center, Taipei, Taiwan or No. 161, MinChuan E.
Rd., Neihu, Taipei 11490, Taiwan, R.O.C.
E-mail: huanghs@ndmctsgh.edu.tw
Fax: þ886-2-87923169
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the telomerase for broad-spectrum cancer therapy [3].
Designing small-molecule drugs with telomerase acitivty is
emerging as an attractive strategy for cancer chemotherapy,
leading to selective inhibition of tumor cell growth [4]. Since
G-quadruplex structure formed by telomere DNA sequences
is not a substrate for telomerase [5, 6], compounds that bind
and stabilize G-quadruplex structures have been considered
as potential inhibitors for telomerase [7]. Indeed, a number
of small molecules that bind to and stabilize the folded
quadruplex were shown to inhibit telomerase activity [8].
The anthracycline antibiotics and related compounds
(Scheme 1) have been used widely as anticancer drugs for
many decades, but their cardiotoxicity limits their clinical
use [9]. The detailed mechanism of how these compounds, all
of which contain an anthraquinone pharmacophore,
remained elusive although they might render their effects
The author contributed equally: Prof. Dr. Jing-Jer Lin,
e-mail: jjlin@ym.edu.tw
102
F.-C. Huang et al.
O
OH
Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
Planar rings
O
R
D
C
OMe O
B
A
X
O
HN
X
O
HN
H
N
for anticancer drugs. The anti-proliferative effect and hTERT
repressing activity of these compounds were determined.
This study enables us to address the issue of how, and to
what extent, the position of side chain substituents affects
telomerase activity.
OH
OH
OH O
O
Me
OH
N
H
NH2
Results and discussion
OH
Doxorubicin (R = OH)
Daunorubicin (R = H)
Chemistry
Mitoxantrone (X = OH)
Ametantrone (X = H)
Scheme 1. Anthracycline antibiotics and related compounds.
through forming complexes with the DNA base pairs or
G-quadruplex [10]. In view of the available information on
SARs, we decided to synthesize anthraquinones containing
two symmetrical and asymmetrical aminoacyl residues in
side chains with spacer linker to explore diverse substituents
in aromatic ring nucleus at positions 1,4-, 1,5-, 2,6-, and 2,7- of
the ring system, respectively. The condensation reactions
with amino acids were accomplished by employing the conventional method of alkylation synthesis. These derivatives
were evaluated for their inhibitory effects on telomerase
activity to identify new molecular or pharmacophore targets
In previous papers we have described a range of synthetic
routes to biological properties of diverse anthraquinonelinked compounds generally symmetrically and asymmetrically substituted in the different positions. The backbone of
these compounds resembles some of the anticancer agents,
mitoxantrone and ametantrone, and was prepared by twostage reaction similar to our previous papers [10]. Our strategy
for achieving this objective is the synthesis of 1,4-, 1,5-, 2,6-,
and 2,7-diamidoanthraquinone derivatives and a number of
acyl chlorides for acylation of aromatic amines A1, A2, B1, B2,
C1 and D1 via N-acylation, respectively. After considerable
effort we were able to develop a process by which 1,4-, 1,5-,
2,6-, and 2,7-disubstituted anthraquinone derivatives can be
synthesized and the preparation involved two-step synthetic
route with appropriate yields (Schemes 2–5): (1) Acylation
O
O
O
Cl
HN
O
HN
R2
N
R1
a
O
HN
NH2
O
Cl
A
NH2
HN
O
O
A1
O
O
R1
c
O
O
O
A3-A9
O
b
O
O
N
R2
HN
O
Cl
O
HN
N
H
d
O
HN
Cl
O
O
A2
H
N
HN
O
O
O
O
O
A10
Reagents and conditions: (a) Chloroacetyl chloride, pyridine, rt, 24 h, 75%; (b) 3-chloropropionyl chloride,
pyridine, rt, 24 h, 44%; (c) L- or D-form of methyl ester of α-amino acids, DIPEA, DMF (A3: R1 = H, R2 = H;
A4: L-form R1 = CH3, R2 = H; A5: D-form R1 = CH3, R2 = H; A6: L-form R1 = CH(CH3)2, R2 = H; A7: D-form
R1 = CH(CH3)2, R2 = H; A8: L-form R1 = CH2CH(CH3)2, R2 = H; A9: R1 = H, R2 = CH3); (d) glycine methyl
ester hydrochloride, DIPEA, DMF.
Scheme 2. Synthesis of compounds A1 and A2 and their disubstituted anthraquinone derivatives A3–A10.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
Telomerase Evaluation and Anti-proliferative Studies
O
O
O
Cl
HN
O
R4
N
HN
O
NH2
Cl
O
H3CO
O
N
R4
O
NH
O
O
B3-B10
O
b
B
OCH3
R3
NH
B1
NH2 O
O
R3
c
a
103
O
O
HN
Cl
O
HN
N
H
d
O
Cl
NH
O
H3CO
H
N
NH
O
OCH3
O
O
O
B2
B11
Reagents and conditions: (a) Chloroacetyl chloride, pyridine, rt, 24 h, 80%; (b) 3-chloropropionyl chloride,
pyridine, rt, 24 h, 45%; (c) L- or D-form of methyl ester of α-amino acids, mini-reactor, 130-150 °C, DIPEA,
DMF (B3: L-form R3 = CH3, R4 = H; B4: D-form R3 = CH3, R4 = H; B5: L-form R3 = CH(CH3)2, R4 = H; B6:
D-form R3 = CH(CH3)2, R4 = H; B7: L-form R3 = CH2CH(CH3)2, R4 = H; B8: R3 = CH2 CH2COOCH3, R4 = H;
B9: R3 = H, R4 = CH3); (d) glycine methyl ester hydrochloride, mini-reactor, 130-150 °C, DIPEA, DMF.
Scheme 3. Synthesis of compounds B1 and B2 and their disubstituted anthraquinone derivatives B3–B11.
O
O
NH2
a
Cl
H2N
H
N
O
N
H
O
Cl
O
O
C1
C
b
O
O
R6
N
H3CO
R5
O
N
H
R5
H
N
O
N
R6
OCH3
O
O
C2-C11
Reagents and conditions: (a) Chloroacetyl chloride, pyridine, rt, 24 h, 80 %; (b) L- or D-form of methyl ester of
α-amino acids, mini-reactor, 130-150 °C, DIPEA, DMF (C2: R5 = H, R6 = H; C3: L-form, R5 = CH3, R6 = H; C4:
D-form, R5 = CH3, R6 = H; C5: L-form, R5 = CH(CH3)2, R6 = H; C6: D-form, R5 = CH(CH3)2, R6 = H; C7:
L-form, R5 = CH2CH(CH3)2, R6 = H; C8: R5 = H, R6 = CH3; C9: S-form, R5 = C6H5, R6 = H; C10: R-form, R5 =
C6H5, R6 = H; C11: S-form, R5 = CH2C6H5, R6 = H).
Scheme 4. Synthesis of compound C1 and its disubstituted anthraquinone derivatives C2–C11.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
O
H2N
NH2
a
H
N
Cl
O
H
N
O
Cl
O
O
O
D1
D
b
R7
H3CO
O
N
H
H
N
O
O
R7
H
N
O
N
H
OCH3
O
O
D2-D9
Reagents and conditions: (a) Chloroacetyl chloride, pyridine, rt, 24 h, 80% ; (b) L- or D-form of methyl ester of
α-amino acids, mini-reactor, 130-150ġ°C, DIPEA, DMF (D2: R7 = H; D3: L-form, R7 = CH3; D4: D-form, R7 =
CH3; D5: L-form, R7 = CH(CH3)2; D6: D-form, R7 = CH(CH3)2; D7: S-form, R7 = C6H5; D8: R-form, R7 = C6H5).
Scheme 5. Synthesis of compound D1 and its disubstituted anthraquinone derivatives D2–D9.
reaction of 1,4-, 1,5-, 2,6-, and 2,7-diaminoanthraquinone
with various acyl chlorides yielded the corresponding bis(v-chloroacylamido) side chain compounds; (2) followed by
nucleophilic displacement of the chloride by alkylation of
amino groups of amino acid esters to produce symmetrical
or asymmetrical aminoacyl residue side chain analogs,
respectively. However, under these reaction conditions significant amounts of the reaction products were isolated along
with the desired reaction products. The quantity of the byproducts isolated was substrate dependant and purification of the
reaction mixtures required tedious recrystallization and
chromatography. Under these conditions, the more effective
acylation catalyst diisopropylethylamine can be achieve the
desired amide linker derivatives with appropriate yields.
The molecular formulas were all determined by HRMS. All
compounds were pure and were characterized by spectroscopic data, as shown in the following Experimental section.
Telomerase inhibitory and hTERT repression activities
Telomere is one of the biological targets of anthraquinonelinked derivatives. With unique structural and electronic
features, these compounds bind and stabilize G-quadruplex
structures formed by telomeric DNA sequences [10]. Indeed,
previously we have identified several anthraquinone-linked
derivatives that showed effective telomerase inhibitory activities. Here we first evaluate the effects of these newly synthesized compounds on telomerase inhibition using PCR-based
telomerase assay, TRAP (telomeric repeat amplification protocol). Under the concentration of 100 mM, only A10 and B11
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
gave profound telomerase inhibitory activity (Fig. 1). The
telomerase IC50 values were estimated to be 10 mM for both
compounds (Fig. 2). To further rule out the possible inhibitory effects of PCR reactions caused by G-quarduplex stabilizers [11], compounds A10 and B11 were added after the
telomerase extension step of the TRAP assays (Fig. 2, TRAPm). A similar dose-dependent pattern was observed for A10 in
both TRAP and TRAP-m assays, suggesting that the inhibitory
effect observed in A10 might be due to the inhibition of Taq
polymerase activity. In contrast, no inhibition of the TRAP-m
assay was observed at concentrations of B11 >100 mM.
Thus, B11 is a potent and selective inhibitor of telomerase
activity.
We also designed and expressed secreted alkaline phosphatase (SEAP) reporter gene under the control of hTERT promoter to monitor the expression of hTERT. The expression
of SEAP in H1299 cells harboring PhTERT-SEAP was used as the
criterion to evaluate if anthraquinone-linked derivatives
inhibited the expression of hTERT in cancer cells [10]. The
level of cell viability in these cells was also determined using
MTT assay. As shown in Table 1, parallel comparison of the
drug-treated cells for the cell proliferation and hTERT expression
revealed that most of the compounds did not show selctive
inhibitory effects on hTERT expression. Only compounds A6,
A8, C3, C8, C9, and D8 affected the SEAP expression without
affecting the proliferation of the treated cells. Among them,
C8 which contains 2-(sarcosine methyl ester)acetamido
groups at 2,6-positions appeared to show the best selectivity
toward repressing hTERT expression. Thus, our results showed
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
Telomerase Evaluation and Anti-proliferative Studies
105
Figure 1. Inhibition of telomerase activity by symmetrical and asymmetrical 1,4-, 1,5-, 2,6-, and 2,7- disubstituted diaminoanthraquinonelinked aminoacyl residue derivatives. TRAP assay was conducted using cell extracts prepared from H1299 cells and 2 mg of extracts were
used in each assay. Extended products were separated on a 10% polyacryamide gel and visualized with SYBER Green staining. The photo
pictures of the results are presented. The concentration of test compounds was 100 mM each. P is positive control and N is negative control.
that these newly designed compounds also selectively
repressed the expression of telomerase. It is also interesting
to note that these compounds did not show strong cytotoxicity toward H1299 cells. The most potent compound C4
which contains 2-(alanine methyl ester)acetamido groups
at 2,6-positions showed a TC50 at 8 mM.
Conclusion
In this investigation, we continue to focus our attention on
the role of our systematically synthesized pharmacophores,
the planar anthraquinone derivatives, and to understand the
pharmacological effects of these compounds. The physical,
Figure 2. Inhibition of telomerase activity by A10 and B11. The effects of A10 (A) or B11 (B) on telomerase activity on PCR reactions were
measured using standard TRAP assays. In standard TRAP assays (left panels), various amounts of compounds were incubated with
telomerase-active cell extracts for 5 min at room temperature before the telomerase extension reactions. In assays for the inhibitory effects
on Taq polymerase, TRAP-m (right panels), indicated amounts of the compounds were added to the TRAP reactions after the telomerase
extension steps. PCR reactions were then conducted as in standard TRAP assays. Telomerase-active cell extracts were prepared from
H1299 cells (P). RNase A-treated extracts were used as negative controls (N). The positions of telomerase ladders are indicated.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
Table 1. Effects of various derivatives on survival of H1299 cells,
repressing hTERT expression, and telomerase activity.
Compounds
A3
A4
A5
A6
A7
A8
A9
A10
B1
B2
B4
B5
B6
B7
B8
B9
B10
B11
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
D2
D3
D4
D5
D6
D7
D8
MTT
(TC50) (mM)
SEAP
(IC50) (mM)
Telomerase
inhibition
(IC50) (mM)
54
48
40
>100
63
>100
56
62
23
42
62
37
51
80
56
37
60
23
78
>100
8
49
39
69
>100
>100
63
12
63
39
65
58
45
>100
>100
44
26
20
73
62
64
41
56
8
62
62
68
77
48
29
49
43
41
54
85
7
47
62
48
45
76
53
5
59
50
60
59
70
>100
65
>100
>100
>100
>100
>100
>100
>100
10
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
ND
ND
TC50: Half maximal toxic concentration, the concentration of a
compound where 50% of its maximal cytotoxic effect is observed;
IC50: Half maximal (50%) inhibitory concentration.
share the same structure of the extended arms with difference in the relative positions of substitutions. Previously, we
had also identified several 1,5-diamido- and 1,5-diamino-substituted anthraquinone derivatives that showed potent telomerase inhibition activities [10]. Thus, two extened
substitutions at the 1,5-positions of anthraquinone derivatives appear to be important structure moieties for the telomerase inhibition activity of these compounds.
Significantly, compounds A6, A8, C6, and D8 showed
repression of hTERT activities without causing severe cytotoxic effects toward cancer cells. The results suggest that
these compounds selectively repressed hTERT expression.
SARs analysis suggested that the 1,4-disubstituted anthraquinones, A6 and A8, may represent an important class of compounds for hTERT repressing activity. However, there is
no other apparent correlation between the hTERT repressing
activity and structure. Thus, the exact mechanism of
how these groups contribute to their activity cannot be
easily elucidated with our current data. Nevertheless, compounds A6, A8, A10, B11, C3, C4, C8, C9, and D8 are
the potential active compounds in all of the target
compounds.
Experimental
Melting points were determined with a Büchi B-545 melting
point apparatus and are uncorrected. All reactions were monitored by TLC (silica gel 60 F254). 1H-NMR: Varian GEMINI-300
(300 MHz); d values are in ppm relative to TMS as an internal
standard. Mass spectra (EI, 70 eV, unless otherwise stated): 6210
Time-of-Flight LC/MS, and Micromass TRIO-2000 (GC/MS). Typical
experiments illustrating the general procedures for the preparation of the anthraquinones are described below.
1,4-Bis(chloroacetamido)anthraquinone (A1)
Product A1 was obtained as yellow powder (yield 75%).
Mp: 2842858C (EtOH) [10]. 1H-NMR (300 MHz, DMSO-d6):
d 4.28 (s, 4H), 7.82–7.86 (m, 2H), 8.31–8.34 (m, 2H), 9.17 (s, 2H),
13.25 (s, 2H).
1,4-Bis(3-chloropropionamido)anthraquinone (A2)
chemical and biological properties of diaminoanthraquinone-linked aminoacyl residue derivatives are greatly
affected by its various substituents of the planar ring. Here
we are interested in examining the effects of these compounds on telomerase activity and hTERT exression in
H1299 cells.
In summary, of these series of compounds analyzed, only
compound B11 showed selective inhibitory effect toward
telomerase activity. Compound A10 might also have telomerase inhibitory activity. However, since our analysis on telomerase activity is a PCR-based reaction, the apparent
inhibitory effect of A10 might be due to its inhibition activity
toward Taq DNA polymerase. Interestingly, both B11 and A10
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Product A2 was obtained as yellow powder (yield 44%).
Mp: 2252268C (EtOH) [10]. 1H-NMR (300 MHz, CDCl3): d 3.01
(t, J ¼ 6.3 Hz, 4H), 3.93 (t, J ¼ 6.6 Hz, 4H), 7.82–7.85 (m, 2H),
8.26–8.29 (m, 2H), 9.17 (s, 2H), 12.26 (s, 2H).
1,4-Bis[2-(glycin methyl ester)acetamido]anthraquinone
(A3)
Compound A1 (0.5 mmol) was dissolved in N,N-dimethylformamide (20 mL) and DIPEA (1 mL, 6 mmol) and glycine methyl
ester hydrochloride (0.37 g, 3 mmol) were added under nitrogen.
The mixture was stirred for 48 h at room temperature. Ice was
added to precipitate out the crude product. The resulting precipitate was collected by filtration, washed with diethyl ether,
and purified by crystallization from ethyl acetate. Product 3 was
obtained as red brown powder (yield 30%). Rf: 0.22 (ethyl acetate/
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
n-hexane, 1:1). Mp: 1641658C (EtOH). 1H-NMR (300 MHz, CDCl3):
d 3.59 (s, 4H), 3.63 (s, 4H), 3.79 (s, 6H), 7.80 (t, J ¼ 6.8 Hz, 2H), 8.29
(t, J ¼ 6.6 Hz, 2H), 9.22 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 50.63,
51.98, 53.51, 117.71, 127.00, 128.82, 133.21, 134.20, 137.47,
171.58 (NCO), 172.52 (CCO), 186.35 (CO). HRMS (ESI) m/z calcd.
for C24H24N4O8 [MþH]þ: 497.1594. Found: 497.1657.
L-1,4-Bis[2-(alanine methyl ester)acetamido]anthraquinone (A4)
Compound A1 (0.5 mmol) was dissolved in N,N-dimethylformamide (20 mL) and DIPEA (1 mL, 6 mmol) and L-alanine methyl
ester hydrochloride (0.42 g, 3 mmol) were added under nitrogen.
The mixture was stirred in mini-reactor for 90 min at
1301508C. Ice was added to precipitate out the crude product.
The resulting precipitate was collected by extract with ethyl
acetate, and purified by crystallization from ethyl acetate/nhexane. Product A4 was obtained as red brown powder (yield
26%). Rf: 0.23 (ethyl acetate/n-hexane, 1:1). Mp: 1491508C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 1.56 (d, J ¼ 6.9 Hz, 6H), 3.33
(d, J ¼ 17.4 Hz, 2H), 3.70 (d, J ¼ 17.7 Hz, 2H), 3.47–3.54
(m, 2H), 3.76 (s, 6H), 7.78–7.80 (m, 2H), 8.26–8.29 (m, 2H), 9.22
(s, 2H), 13.25 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 19.00, 46.16,
52.14, 56.87, 117.76, 126.93, 128.75, 133.25, 134.15, 137.36,
171.89 (NCO), 175.76 (CCO), 186.22 (CO). HRMS (ESI) m/z calcd.
for C26H28N4O8 [MþH]þ: 525.1907. Found: 525.1974.
D-1,4-Bis[2-(alanine methyl ester)acetamido]anthraquinone (A5)
Product A5 was obtained as red brown powder (yield 33%).
Rf: 0.23 (ethyl acetate/n-hexane, 1:1). Mp: 1491508C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 1.56 (d, J ¼ 6.9 Hz, 6H), 3.32 (d,
J ¼ 17.4 Hz, 2H), 3.70 (d, J ¼ 17.7 Hz, 2H), 3.46–3.53 (m, 2H),
3.77 (s, 6H), 7.78–7.80 (m, 2H), 8.26–8.29 (m, 2H), 9.22 (s, 2H),
13.26 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 18.99, 46.89, 52.12,
56.86, 117.68, 126.89, 128.69, 133.20, 134.12, 137.40, 171.94
(NCO), 175.74 (CCO), 186.15 (CO). HRMS (ESI) m/z calcd.
for C26H28N4O8 [MþH]þ: 525.1907. Found: 525.1967.
L-1,4-Bis[2-(valine methyl ester)acetamido]anthraquinone (A6)
Product A6 was obtained as red brown powder (yield 30%). Rf:
0.36 (ethyl acetate/n-hexane, 1:1). Mp: 1801818C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.08–1.15 (m, 12H), 2.15–2.21 (m, 2H), 3.17 (d,
J ¼ 5.1 Hz, 2H), 3.24 (d, J ¼ 7.4 Hz, 2H), 3.70 (d, J ¼ 17.1 Hz, 2H),
3.76 (s, 6H), 7.77–7.80 (m, 2H), 8.21–8.24 (m, 2H), 9.17 (s, 2H),
13.09 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 18.53, 19.21, 31.73,
51.73, 53.18, 67.83, 118.15, 126.94, 128.97, 133.41, 134.18,
137.43, 171.77 (NCO), 174.86 (CCO), 186.22 (CO). HRMS (ESI) m/z
calcd. for C30H36N4O8 [MþH]þ: 581.2533. Found: 581.2603.
D-1,4-Bis[2-(valine methyl ester)acetamido]anthraquinone (A7)
Product A7 was obtained as red brown powder (yield 43%). Rf:
0.36 (ethyl acetate/n-hexane, 1:1). Mp: 1801818C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.10–1.17 (m, 12H), 2.22–2.28 (m, 2H), 3.27 (d,
J ¼ 5.4 Hz, 2H), 3.36 (d, J ¼ 17.1 Hz, 2H), 3.71 (t, J ¼ 7.8 Hz, 2H),
3.78 (s, 6H), 7.78–7.80 (m, 2H), 8.21–8.24 (m, 2H), 9.15 (s, 2H),
13.07 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 18.50, 19.16, 31.68,
51.66, 53.12, 67.79, 117.95, 126.85, 128.79, 133.28, 134.10,
137.34, 171.70 (NCO), 174.80 (CCO), 186.03 (CO). HRMS (ESI) m/z
calcd. for C30H36N4O8 [MþH]þ: 581.2533. Found: 581.2596.
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L-1,4-Bis[2-(leucine methyl ester)acetamido]anthraquinone (A8)
Product A8 was obtained as red brown powder (yield 39%).
Rf: 0.34 (ethyl acetate/n-hexane, 1:1). Mp: 2002018C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 0.92–0.98 (m, 12H), 1.58–2.03
(m, 6H), 3.27 (d, J ¼ 17.1 Hz, 2H), 3.69 (d, J ¼ 17.4 Hz, 2H),
3.41 (t, 2H), 3.75 (s, 6H), 7.79–7.82 (m, 2H), 8.23–8.26 (m, 2H),
9.21 (s, 2H), 13.16 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 22.43, 22.79,
24.65, 42.66, 51.87, 52.46, 60.26, 117.76, 126.82, 128.81, 133.38,
134.15, 137.44, 171.81 (NCO), 175.79 (CCO), 186.11 (CO). HRMS
(ESI) m/z calcd. for C32H40N4O8 [MþH]þ: 609.2846. Found:
609.2909.
1,4-Bis[2-(sarcosine methyl ester)acetamido]anthraquinone (A9)
Product A9 was obtained as red brown powder (yield 45%).
Rf: 0.43 (ethyl acetate/n-hexane, 1:1). Mp: 1501518C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 2.67 (s, 6H), 3.53 (s, 4H), 3.64
(s, 4H), 3.75 (s, 6H), 7.77–7.80 (m, 2H), 8.27–8.30 (m, 2H), 9.20
(s, 2H), 13.21 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 42.85, 51.51,
57.95, 61.31, 118.00, 126.99, 128.77, 133.30, 134.02, 137.36,
171.07 (NCO), 171.32 (CCO), 186.21 (CO). ESI-MS m/z: 525.3
[MþH]þ.
1,4-Bis[2-(glycin methyl ester)propionamido]anthraquinone (A10)
Product A10 was obtained as red brown powder (yield 23%). Rf:
0.22 (ethyl acetate/n-hexane, 1:1). Mp: 1551568C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 2.50 (s, 2H), 2.73 (t, 4H, –CH2–), 3.07 (t, 4H,
–CH2–), 3.51 (s, 4H, –CH2–), 3.73 (s, 6H, –OCH3–), 7.79–7.82 (m,
2H), 8.24–8.67 (m, 2H), 9.13 (s, 2H) 12.59 (s, 2H). 13C-NMR (75 MHz,
CDCl3): d 38.91, 45.13, 50.76, 51.82, 116.98, 127.09, 129.21,
133.32, 134.43, 138.26, 171.57 (NCO), 172.73 (CCO), 186.85
(CO). HRMS (ESI) m/z calcd. for C26H28N4O8 [MþH]þ: 525.1907.
Found: 525.1960.
L-1,5-Bis[2-(alanine methyl ester)acetamido]anthraquinone (B3)
Product B3 was obtained as brown powder (yield 40%). Rf: 0.26
(ethyl acetate/n-hexane, 2:1). Mp: 1411428C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.56 (d, J ¼ 7.2 Hz, 6H), 3.32 (d,
J ¼ 17.7 Hz, 2H), 3.70 (d, J ¼ 17.4 Hz, 2H), 3.50 (dd, J ¼ 15.0,
7.2 Hz, 2H), 3.76 (s, 6H), 7.75 (t, J ¼ 8.1 Hz, 2H), 8.05 (d,
J ¼ 7.5 Hz, 2H), 9.20 (d, J ¼ 8.7 Hz, 2H), 13.09 (s, 2H). 13C-NMR
(75 MHz, CDCl3): d 18.99, 52.01, 52.05, 56.89, 117.60, 122.60,
125.95, 134.53, 135.44, 140.76, 172.20 (NCO), 175.73 (CCO),
185.77 (CO). ESI-MS m/z: 525.2 [MþH]þ.
D-1,5-Bis[2-(alanine methyl ester)acetamido]anthraquinone (B4)
Product B4 was obtained as yellow powder (yield 45%).
Rf: 0.26 (ethyl acetate/n-hexane, 2:1). Mp: 1431448C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 1.57 (d, J ¼ 6.9 Hz, 6H), 3.32
(d, J ¼ 17.7 Hz, 2H), 3.71 (d, J ¼ 17.4 Hz, 2H), 3.52 (dd,
J ¼ 14.1, 7.2 Hz, 2H), 3.77 (s, 6H, –OCH3), 7.75 (t, J ¼ 9.0 Hz,
2H), 8.05 (d, J ¼ 7.5 Hz, 2H), 9.20 (d, J ¼ 8.4 Hz, 2H),
13.10 (s, 2H). 13C-NMR (75 MHz, CDCl3): d 18.99, 52.01,
52.05, 56.89, 117.61, 122.60, 125.95, 134.54, 135.44, 140.76,
172.18 (NCO), 175.72 (CCO), 185.77 (CO). ESI-MS m/z: 525.2
[MþH]þ.
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F.-C. Huang et al.
L-1,5-Bis[2-(valine methyl ester)acetamido]anthraquinone (B5)
Product B5 was obtained as yellow brown powder (yield 35%).
Rf: 0.45 (ethyl acetate/n-hexane, 2:1). Mp: 1251268C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 1.09–1.16 (m, 12H, –CH3), 2.19–
2.23 (m, 2H, –CH–), 3.22 (d, J ¼ 5.1 Hz, 2H, –CH–), 3.31
(d, J ¼ 16.8 Hz, 2H, –CH2–), 3.74 (d, J ¼ 18.5 Hz, 2H, –CH2–),
3.77 (s, 6H, –OCH3), 7.75 (t, J ¼ 8.1 Hz, 2H), 8.01 (d, J ¼ 7.8 Hz,
2H), 9.15 (d, J ¼ 8.4 Hz, 2H), 12.92 (s, 2H, Ar–NH–). 13C-NMR
(75 MHz, CDCl3): d 18.48, 19.14, 31.67, 51.68, 53.10, 67.83,
117.76, 122.57, 126.10, 134.61, 135.41, 140.72, 171.97 (NCO),
174.78 (CCO), 185.52 (CO). ESI-MS m/z: 581.3 [MþH]þ.
D-1,5-Bis[2-(valine methyl ester)acetamido]anthraquinone (B6)
Product B6 was obtained as yellow brown powder (yield 41%).
Rf: 0.45 (ethyl acetate/n-hexane, 2:1). Mp: 1261278C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 1.08–1.19 (m, 12H, –CH3), 2.17–
2.23 (m, 2H, –CH–), 3.19 (d, J ¼ 5.1Hz, 2H, –CH–), 3.28
(d, J ¼ 17.4 Hz, 2H, –CH2–), 3.72 (d, J ¼ 17.4 Hz, 2H, –CH2–),
3.76 (s, 6H, –OCH3), 7.74 (t, J ¼ 8.1 Hz, 2H), 8.01 (d, J ¼ 7.5 Hz,
2H), 9.15 (d, J ¼ 8.4 Hz, 2H), 12.92 (s, 2H, Ar–NH–). 13C-NMR
(75 MHz, CDCl3): d 18.51, 19.17, 31.70, 51.72, 53.14, 67.85,
117.88, 122.65, 126.19, 134.71, 135.47, 140.77, 171.98 (NCO),
174.80 (CCO), 185.66 (CO). ESI-MS m/z: 581.3 [MþH]þ.
L-1,5-Bis[2-(leucine methyl ester)acetamido]anthraquinone (B7)
Product B7 was obtained as yellow brown powder (yield 35%).
Rf: 0.47 (ethyl acetate/n-hexane, 1:1). Mp: 1491508C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 0.920.98 (m, 12H, –CH3), 1.62–
2.04 (m, 6H, –CH2–CH–), 3.29 (d, J ¼ 17.1 Hz, 2H, –CH2–), 3.71
(d, J ¼ 17.4 Hz, 2H, –CH2–), 3.42 (t, J ¼ 6.6 Hz, 2H, –CH–), 3.76
(s, 6H, –OCH3), 7.76 (t, J ¼ 8.1 Hz, 2H), 8.01 (d, J ¼ 7.8 Hz, 2H),
9.18 (d, J ¼ 8.7 Hz, 2H), 12.97 (s, 2H, Ar–NH–). 13C-NMR (75 MHz,
CDCl3): d 22.43, 22.76, 24.63, 42.66, 46.12, 51.88, 52.42, 60.29,
117.76, 122.48, 126.07, 134.69, 135.52, 140.83, 172.02 (NCO),
175.77 (CCO), 185.64 (CO). ESI-MS m/z: 609.4 [MþH]þ.
1,5-Bis[2-(glutamic acid dimethyl ester)acetamido]anthraquinone (B8)
Product B8 was obtained as red brown powder (yield 38%). Rf: 0.32
(ethyl acetate/n-hexane, 2:1). Mp: 1511528C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 2.11–2.25 (m, 4H, –CH2–), 2.71–2.86
(m, 4H, –CH2–), 3.35 (d, J ¼ 17.4 Hz, 2H, –CH2–), 3.65
(d, J ¼ 17.1 Hz, 2H, –CH2–), 3.50 (t, J ¼ 6.0 Hz, 2H, –CH–), 3.62
(s, 6H, –OCH3), 3.78 (s, 6H, –OCH3), 7.74 (t, J ¼ 9.6 Hz, 2H), 8.03
(d, J ¼ 7.5 Hz, 2H), 9.16 (d, J ¼ 8.7 Hz, 2H), 12.97 (s, 2H, Ar–NH–).
13
C-NMR (75 MHz, CDCl3): d 28.34, 30.08, 51.58, 52.15, 52.62,
60.98, 117.64, 122.74, 126.03, 134.58, 135.55, 140.78, 171.78
(NCO), 173.50 (CCO), 174.69 (CCO), 185.73 (CO). ESI-MS m/z:
669.4 [MþH]þ.
1,5-Bis[2-(sarcosine methyl ester)acetamido]anthraquinone (B9)
Product B9 was obtained as red brown powder (yield 55%). Rf: 0.48
(ethyl acetate/n-hexane, 1:1). Mp: 1861878C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 2.67 (s, 6H, –N–CH3), 3.53 (s, 4H, –CH2–),
3.64 (s, 4H, –CH2–), 3.75 (s, 6H, –OCH3), 7.76 (t, J ¼ 8.4 Hz, 2H),
8.07 (d, J ¼ 6.6 Hz, 2H), 9.17 (d, J ¼ 7.5 Hz, 2H), 13.05 (s, 2H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
Ar–NH–). 13C-NMR (75 MHz, CDCl3): d 22.42, 22.75, 24.63, 42.65,
51.87, 52.41, 60.29, 117.76, 122.48, 126.07, 134.68, 135.51,
140.82, 172.02 (NCO), 175.76 (CCO), 185.64 (CO). ESI-MS m/z:
525.3 [MþH]þ.
1,5-Bis[2-(ß-alanine methyl ester)acetamido]anthraquinone (B10)
Product B10 was obtained as red brown powder (yield 40%).
Rf: 0.29 (ethyl acetate/n-hexane, 2:1). Mp: 1471488C (EtOH).
1H-NMR (300 MHz, CDCl3): d 2.79 (t, J ¼ 6.0 Hz, 4H, –NCH2–),
3.02 (t, J ¼ 6.7 Hz, 4H, –CH2CO–), 3.55 (s, 4H, –CH2–), 3.72 (s, 6H,
–OCH3), 7.76 (t, J ¼ 8.4 Hz, 2H), 8.09 (d, J ¼ 9.0 Hz, 2H), 9.22 (d,
J ¼ 9.6 Hz, 2H), 13.09 (s, 2H, Ar–NH–). 13C-NMR (75 MHz, CDCl3):
d 34.28, 45.37, 46.13, 51.73, 53.71, 64.45, 117.35, 122.61, 126.08,
134.68, 135.52, 140.89, 172.47 (NCO), 173.11 (CCO), 185.83 (CO).
ESI-MS m/z: 525.3 [MþH]þ.
1,5-Bis[2-(glycin methyl ester)propionamido]anthraquinone (B11)
Product B11 was obtained as red brown powder (yield 25%).
Rf: 0.32 (ethyl acetate/n-hexane, 2:1). Mp: 1501518C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 2.74 (t, J ¼ 6.3 Hz, 4H, –CH2N–),
3.08 (t, J ¼ 6.3 Hz, 4H, –COCH2–), 3.51 (s, 4H, –CH2–), 3.73 (s,
6H, –OCH3), 7.77 (t, J ¼ 7.8 Hz, 2H), 8.03 (d, J ¼ 7.5 Hz, 2H), 9.13
(d, J ¼ 8.4 Hz, 2H), 12.35 (s, 2H, Ar–NH–). 13C-NMR (75 MHz,
CDCl3): d 38.91, 45.13, 50.76, 51.82, 116.98, 127.09, 129.21,
133.32, 134.43, 138.26, 171.57 (NCO), 172.73 (CCO), 186.85
(CO). HRMS (ESI) m/z calcd. for C26H28N4O8 [MþH]þ: 525.1907.
Found: 525.1964.
2,6-Bis(chloroacetamido)anthraquinone (C1)
Yield: 75%. Mp: 3233248C (EtOH) [12]. 1H-NMR (300 MHz,
DMSO-d6): d 4.29 (s, 4H, –CH2–), 7.99 (d, J ¼ 6.9 Hz, 2H, H-4,8),
8.12 (d, J ¼ 8.4 Hz, 2H, H-3,7), 8.36 (s, 2H, H-1,5), 10.88 (s, 2H,
–NH–).
2,6-Bis[2-(glycin methyl ester)acetamido]anthraquinone (C2)
Product C2 was obtained as yellow powder (yield 26%). Rf: 0.2
(ethyl acetate/n-hexane, 1:1). Mp: 1771788C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 3.49 (s, 4H, –CH2–), 3.53 (s, 4H, –CH2–),
3.77 (s, 6H, –OCH3–), 8.15 (s, 2H, H-1,5), 8.27 (d, J ¼ 8.4 Hz, 2H,
H-4,8), 8.36 (d, J ¼ 6.6 Hz, 2H, H-3,7), 9.86 (s, 2H, NH). 13C-NMR
(75 MHz, CDCl3): d 50.63, 51.98, 53.51, 117.71, 127.00, 128.82,
133.21, 134.20, 137.47, 171.58 (NCO), 172.52 (CCO), 186.35 (CO).
HRMS (ESI) m/z calcd. for C24H24N4O8 [MþH]þ: 497.1594. Found:
497.1659.
L-2,6-Bis[2-(alanine methyl ester)acetamido]anthraquinone (C3)
Product C3 was obtained as yellow powder (yield 36%). Rf: 0.28
(ethyl acetate/n-hexane, 1:1). Mp: 1311328C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.43 (d, J ¼ 6.9 Hz, 6H, –CH3), 3.33 (d,
J ¼ 17.4 Hz, 2H, –CH2–), 3.54 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.41–
3.48 (m, 2H, –CH–), 3.77 (s, 6H, –CH3), 8.11 (s, 2H, H-1,5), 8.29 (d,
J ¼ 8.7 Hz, 2H, H-4,8), 8.39 (d, J ¼ 9.0 Hz, 2H, H-3,7), 9.80 (s, 2H,
–NH–). 13C-NMR (75 MHz, CDCl3): d 19.11, 51.66, 52.34, 57.17,
116.57, 123.79, 129.27, 129.34, 134.86, 143.11, 170.10 (NCO),
175.09 (CCO), 181.79 (CO). HRMS (ESI) m/z calcd. for
C26H28N4O8 [MþH] þ: 525.1907. Found: 525.1974.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
D-2,6-Bis[2-(alanine methyl ester)acetamido]anthraquinone (C4)
Product C4 was obtained as yellow powder (yield 33%). Rf: 0.28
(ethyl acetate/n-hexane, 1:1). Mp: 1311328C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.44 (d, J ¼ 6.9 Hz, 6H, –CH3), 3.34 (d,
J ¼ 17.4 Hz, 2H, –CH2–), 3.55 (d, J ¼ 17.4 Hz, 2H, –CH2–), 3.46
(dd, J ¼ 11.4, 6.3 Hz, 2H, –CH–), 3.78 (s, 6H, –CH3), 8.12 (s, 2H, H1,5), 8.29 (d, J ¼ 9.0 Hz, 2H, H-4,8), 8.39 (d, J ¼ 9.0 Hz, 2H, H-3,7),
9.81 (s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 19.09, 51.65,
52.32, 57.15, 116.53, 123.75, 129.22, 129.28, 134.82, 143.09,
170.11 (NCO), 175.10 (CCO), 181.74 (CO). HRMS (ESI) m/z calcd.
for C26H28N4O8 [MþH] þ: 525.1907. Found: 525.1964.
L-2,6-Bis[2-(valine methyl ester)acetamido]anthraquinone (C5)
Product C5 was obtained as yellow powder (yield 41%). Rf: 0.17
(ethyl acetate/n-hexane, 1:1). Mp: 2002018C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.03–1.10 (m, 12H, –CH3), 2.07–2.13 (m,
2H, –CH–), 3.10 (d, J ¼ 5.4 Hz, 2H, –CH–), 3.19 (d, J ¼ 17.7 Hz,
2H –CH2–), 3.60 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.76 (s, 6H, –CH3),
8.11 (s, 2H, H-1,4), 8.27 (d, J ¼ 8.4 Hz, 2H, H-4,8), 8.33 (d,
J ¼ 8.7 Hz, 2H, H-3,7), 9.76 (s, 2H, –NH–). 13C-NMR (75 MHz,
CDCl3): d 18.20, 19.69, 31.44, 52.02, 52.15, 67.83, 116.31,
123.62, 129.24, 134.83, 143.02, 170.06 (NCO), 174.55 (CCO),
181.61 (CO). HRMS (ESI) m/z calcd. for C30H36N4O8 [MþH]þ:
581.2533. Found: 581.2595.
D-2,6-Bis[2-(valine methyl ester)acetamido]anthraquinone (C6)
Product C6 was obtained as yellow powder (yield 43%). Rf: 0.17
(ethyl acetate/n-hexane, 1:1). Mp: 2002018C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.03–1.10 (m, 12H, –CH3), 2.07–2.13 (m,
2H, –CH–), 3.10 (d, J ¼ 5.4 Hz, 2H, –CH–), 3.19 (d, J ¼ 17.7 Hz,
2H, –CH2–), 3.60 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.76 (s, 6H, –CH3),
8.11 (s, 2H), 8.27 (d, J ¼ 8.4 Hz, 2H), 8.33 (d, J ¼ 8.7 Hz, 2H), 9.76
(s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 18.16, 19.65, 31.41,
51.99, 52.11, 67.79, 116.26, 123.56, 129.18, 134.77, 142.98, 170.04
(NCO), 174.52 (CCO), 181.54 (CO). HRMS (ESI) m/z calcd
for C30H36N4O8 [MþH]þ: 581.2533. Found: 581.2587.
L-2,6-Bis[2-(leucine methyl ester)acetamido]anthraquinone (C7)
Product C7 was obtained as yellowish brown powder (yield 39%).
Rf: 0.26 (ethyl acetate/n-hexane, 1:1). Mp: 1791808C (EtOH). 1HNMR (300 MHz, CDCl3): d 0.96–1.02 (m, 12H, –CH3), 1.55–1.92 (m,
6H, –CH2–CH–), 3.25 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.56 (d,
J ¼ 17.7 Hz, 2H, –CH2–), 3.35 (t, J ¼ 6.6 Hz, 2H, –CH–), 3.75 (s,
6H, –OCH3), 8.10 (s, 2H), 8.27 (d, J ¼ 8.7 Hz, 2H), 8.34 (d,
J ¼ 8.4 Hz, 2H), 9.77 (s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d
21.85, 22.92, 25.11, 42.49, 51.80, 52.15, 60.39, 116.37, 123.60,
129.18, 129.21, 134.79, 142.99, 170.05 (NCO), 175.37 (CCO), 181.61
(CO). HRMS (ESI) m/z calcd. for C32H40N4O8 [MþH]þ: 609.2846.
Found: 609.2911.
2,6-Bis[2-(sarcosine methyl ester)acetamido]anthraquinone (C8)
Product C8 was obtained as yellow powder (yield 58%). Rf: 0.23
(ethyl acetate/n-hexane, 1:1). Mp: 1451468C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 2.55 (s, 6H, -N–CH3), 3.36 (s, 4H, –CH2–),
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Telomerase Evaluation and Anti-proliferative Studies
109
3.46 (s, 4H, –CH2–), 3.79 (s, 6H,–OCH3), 8.20 (s, 2H, H-1,4), 8.28
(d, J ¼ 8.7 Hz, 2H), 8.37 (d, J ¼ 8.4 Hz, 2H), 10.03 (s, 2H, –NH–).
13
C-NMR (75 MHz, CDCl3): d 51.44, 52.71, 65.75, 116.58, 123.73,
127.43, 129.18, 134.70, 137.10, 142.97, 169.89 (NCO), 172.82
(CCO), 181.64 (CO). ESI-MS m/z: 525.3 [MþH]þ.
S-2,6-Bis[2-(phenylglycin methyl ester)acetamido]anthraquinone (C9)
Product C9 was obtained as yellowish brown powder (yield 44%).
Rf: 0.23 (ethyl acetate/n-hexane, 1:1). Mp: 2102118C (EtOH).
1
H-NMR (300 MHz, CDCl3): d 3.42 (d, J ¼ 17.4 Hz, 2H, –CH2–),
3.51 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.75 (s, 6H, –CH3), 4.43 (s, 2H,
–CH–), 7.39 (s, 10H, -C6H5), 8.06 (s, 2H), 8.27 (d, J ¼ 4.2 Hz, 4H),
9.68 (s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 51.46, 52.73,
65.79, 116.60, 123.76, 127.43, 128.89, 129.18, 129.25, 134.76,
137.08, 142.99, 169.83 (NCO), 172.79 (CCO), 181.69 (CO). HRMS
(ESI) m/z calcd. for C36H32N4O8 [MþH]þ: 649.2220. Found:
649.2307.
R-2,6-Bis[2-(phenylglycin methyl ester)acetamido]anthraquinone (C10)
Product C10 was obtained as yellow powder (yield 35%). Rf: 0.22
(ethyl acetate/n-hexane, 1:1). Mp: 2022038C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 3.39 (d, J ¼ 17.1 Hz, 2H, –CH2–), 3.48 (d,
J ¼ 18.0 Hz, 2H, –CH2–), 3.73 (s, 6H, –CH3), 4.41 (s, 2H, –CH–),
7.37 (s, 10H, -C6H5), 8.04 (s, 2H), 8.24 (d, J ¼ 3.6 Hz, 4H), 9.66 (s,
2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 51.32, 51.37, 53.46,
116.68, 123.08, 128.55, 128.77, 129.02, 129.12, 134.53, 137.39,
142.66, 169.58 (NCO), 174.34 (CCO), 181.54 (CO). HRMS (ESI) m/z
calcd. for C36H32N4O8 [MþH]þ: 649.2220. Found: 649.2263.
2,6-Bis[2-(phenylalanine methyl ester)acetamido]anthraquinone (C11)
Product C11 was obtained as yellowish brown powder (yield
25%). Rf: 0.32 (ethyl acetate/n-hexane, 1:1). Mp: 1971988C
(EtOH). 1H-NMR (300 MHz, CDCl3): d 2.75 (t, J ¼ 12.9 Hz, 2H,
–CH–), 3.16 (d, J ¼ 18.0 Hz, 2H, –CH2–), 3.55 (d, J ¼ 18.0 Hz,
2H, –CH2–), 3.79 (s, 6H, –CH3), 7.27–7.42 (m, 10H, -C6H5), 7.72
(s, 2H), 7.93 (d, J ¼ 8.7 Hz, 2H), 8.19 (d, J ¼ 8.7 Hz, 2H), 9.08 (s, 2H,
–NH–). 13C-NMR (75 MHz, CDCl3): d 39.51, 51.51, 52.37, 63.46,
116.67, 123.68, 127.55, 129.02, 134.52, 137.19, 143.66, 169.89
(NCO), 174.40 (CCO), 181.54 (CO). HRMS (ESI) m/z calcd.
for C38H36N4O8 [MþH]þ: 677.2533. Found: 677.2593.
2,7-Bis(chloroacetamido)anthraquinone (D1)
Yield: 60%. Mp: 2662678C (EtOH) [10]. 1H-NMR (300 MHz,
DMSO-d6): d 4.33 (s, 4H, –CH2–), 8.02 (dd, J ¼ 8.7, 2.4 Hz, 2H),
8.13 (d, J ¼ 8.7 Hz, 2H), 8.40 (d, J ¼ 2.1 Hz, 2H), 10.90 (s, 2H,
–NH–).
2,7-Bis[2-(glycin methyl ester)acetamido]anthraquinone (D2)
Product D2 was obtained as yellow powder (yield 26%). Rf: 0. 23
(ethyl acetate/n-hexane, 2:1). Mp: 1471488C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 3.49 (s, 4H, –CH2–), 3.54 (s, 4H, –CH2–),
3.77 (s, 6H, –OCH3), 8.19 (s, 2H, H-1,8), 8.28 (d, J ¼ 8.1 Hz, 2H),
8.32 (d, J ¼ 9.0 Hz, 2H), 9.84 (s, 2H, –NH–). 13C-NMR (75 MHz,
CDCl3): d 50.63, 51.98, 53.51, 117.71, 127.00, 128.82, 133.21,
134.20, 137.47, 171.58 (NCO), 172.52 (CCO), 186.35 (CO). HRMS
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110
F.-C. Huang et al.
(ESI) m/z calcd. for C24H24N4O8 [MþH]þ: 497.1594. Found:
497.1654.
L-2,7-Bis[2-(alanine methyl ester)acetamido]anthraquinone (D3)
Product D3 was obtained as yellow powder (yield 30%). Rf: 0. 28
(ethyl acetate/n-hexane, 2:1). Mp: 1471488C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.43 (d, J ¼ 6.9 Hz, 6H, –CH3), 3.33 (d,
J ¼ 17.4 Hz, 2H, –CH2–), 3.54 (d, J ¼ 17.1 Hz, 2H, –CH2–), 3.45
(dd, J ¼ 13.8, 6.9 Hz, 2H, –CH–), 3.76 (s, 6H, –CH3), 8.15 (s, 2H, H1,8), 8.28 (d, J ¼ 8.4 Hz, 2H), 8.33 (d, J ¼ 8.4Hz, 2H), 9.78 (s, 2H,
–NH–). 13C-NMR (75 MHz, CDCl3): d 19.08, 51.63, 52.31, 57.13,
116.52, 124.14, 129.19, 129.35, 134.60, 142.73, 170.12 (NCO),
175.11 (CCO), 182.68 (CO). HRMS (ESI) m/z calcd.
for C26H28N4O8 [MþH]þ: 525.1907. Found: 525.1975.
D-2,7-Bis[2-(alanine methyl ester)acetamido]anthraquinone (D4)
Product D4 was obtained as yellow powder (yield 25%). Rf: 0. 28
(ethyl acetate/n-hexane, 1:2). Mp: 1451468C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.44 (d, J ¼ 6.6 Hz, 6H, –CH3), 3.34 (d,
J ¼ 17.7 Hz, 2H, –CH2–), 3.56 (d, J ¼ 17.1 Hz, 2H, –CH2–), 3.46
(dd, J ¼ 13.8, 6.9 Hz, 2H, –CH–), 3.78 (s, 6H, –CH3), 8.16 (s, 2H, H1,8), 8.30 (d, J ¼ 8.4 Hz, 2H, H-4,5), 8.35 (d, J ¼ 8.7 Hz, 2H, H-3,6),
9.79 (s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 19.08, 51.63,
52.31, 57.12, 116.50, 124.11, 129.17, 129.33, 134.59, 142.73,
170.13 (NCO), 175.11 (CCO), 182.65 (CO). HRMS (ESI) m/z calcd.
for C26H28N4O8 [MþH]þ: 525.1907. Found: 525.1963.
L-2,7-Bis[2-(valine methyl ester)acetamido]anthraquinone (D5)
Product D5 was obtained as yellow powder (yield 45%). Rf: 0.30
(ethyl acetate/n-hexane, 2:1). Mp: 1911928C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.04–1.11 (m, 12H, –CH3), 2.13 (d,
J ¼ 5.4 Hz, 2H, –CH–), 3.12 (s, 2H, –CH–), 3.22 (d, J ¼ 17.7 Hz,
2H –CH2–), 3.62 (d, J ¼ 17.4 Hz, 2H, –CH2–), 3.77 (s, 6H, –CH3),
8.15 (s, 2H, H-1,8), 8.28 (s, 4H, H-4,3,5,6), 9.78 (s, 2H, –NH–). 13CNMR (75 MHz, CDCl3): d 18.16, 19.68, 31.42, 52.02, 52.12, 67.81,
116.28, 123.95, 129.17, 129.32, 134.64, 142.67, 170.53 (NCO),
174.55 (CCO), 182.49 (CO). HRMS (ESI) m/z calcd.
for C30H36N4O8 [MþH]þ: 581.2533. Found: 581.2608.
D-2,7-Bis[2-(valine methyl ester)acetamido]anthraquinone (D6)
Product D6 was obtained as yellow powder (yield 40%). Rf: 0. 30
(ethyl acetate/n-hexane, 2:1). Mp: 1921938C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 1.04–1.11 (m, 12H, –CH3), 2.12 (s, 2H,
–CH–), 3.13 (s, 2H, –CH–), 3.23 (d, J ¼ 19.2 Hz, 2H –CH2–), 3.63
(d, J ¼ 18.6 Hz, 2H, –CH2–), 3.77 (s, 6H, –CH3), 8.14 (s, 2H, H-1,8),
8.27 (s, 4H, H-4,3,5,6), 9.79 (s, 2H, –NH–). 13C-NMR (75 MHz,
CDCl3): d 17.55, 18.89, 30.82, 51.30, 51.60, 67.36, 115.89,
123.60, 128.71, 129.05, 134.38, 142.32, 169.36 (NCO), 173.94
(CCO), 182.16 (CO). HRMS (ESI) m/z calcd. for C30H36N4O8
[MþH]þ: 581.2533. Found: 581.2599.
S-2,7-Bis[2-(phenylglycin methyl ester) acetamido]anthraquinone (D7)
Product D7 was obtained as yellowish brown powder (yield 32%).
Rf: 0. 32 (ethyl acetate/n-hexane, 2:1). Mp: 1601618C (EtOH).
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
1
H-NMR (300 MHz, CDCl3): d 3.41 (d, J ¼ 17.1 Hz, 2H, –CH2–),
3.50 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.75 (s, 6H, –CH3), 4.43 (s, 2H,
–CH–), 7.39 (s, 10H, -C6H5), 8.08 (s, 2H, H-1,8), 8.25 (s, 4H, H4,3,5,6), 9.66 (s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 51.46,
52.73, 65.79, 116.60, 123.76, 127.43, 129.18, 134.76, 137.08,
142.99, 169.83 (NCO), 172.79 (CCO), 181.69 (CO). HRMS (ESI) m/z
calcd. for C36H32N4O8 [MþH]þ: 649.2220. Found: 649.2272.
R-2,7-Bis[2-(phenylglycin methyl ester)
acetamido]anthraquinone (D8)
Product D8 was obtained as yellow powder (yield 35%). Rf: 0. 32
(ethyl acetate/n-hexane, 2:1). Mp: 1651668C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 3.43 (d, J ¼ 6.0 Hz, 2H –CH2–), 3.51 (d,
J ¼ 16.5 Hz, 2H, –CH2–), 3.75 (s, 6H, –CH3), 4.44 (s, 2H, –CH–),
7.40 (s, 10H, -C6H5), 8.10 (s, 2H, H-1,8), 8.25 (s, 4H, H-4,3,5,6), 9.68
(s, 2H, –NH–). 13C-NMR (75 MHz, CDCl3): d 51.43, 52.72, 65.75,
116.57, 124.10, 127.42, 129.07, 134.53, 137.11, 142.65, 169.89
(NCO), 172.83 (CCO), 182.54 (CO). HRMS (ESI) m/z calcd.
for C36H32N4O8 [MþH]þ: 649.2220. Found: 649.2285.
2,7-Bis[2-(phenylalanine methyl ester)
acetamido]anthraquinone (D9)
Product D9 was obtained as yellow powder (yield 42%). Rf: 0. 35
(ethyl acetate/n-hexane, 2:1). Mp: 1911928C (EtOH). 1H-NMR
(300 MHz, CDCl3): d 2.76 (t, J ¼ 10.2 Hz, 2H –CH–), 3.18 (d,
J ¼ 18.0 Hz, 2H –CH2–), 3.57 (d, J ¼ 17.7 Hz, 2H, –CH2–), 3.81
(s, 6H, –CH3), 7.28–7.43 (m, 10H, -C6H5), 7.78 (s, 2H, H-1,8), 7.88 (d,
J ¼ 6.3 Hz, 2H, H-4,5), 8.18 (d, J ¼ 8.4 Hz, 2H, H-3,6), 9.09 (s, 2H,
–NH–). 13C-NMR (75 MHz, CDCl3): d 39.56, 51.51, 52.38, 63.49,
116.65, 123.94, 127.57, 129.06, 134.46, 137.24, 142.40, 169.86
(NCO), 174.41 (CCO), 182.20 (CO). HRMS (ESI) m/z calcd.
for C38H36N4O8 [MþH] þ: 677.2533. Found: 677.2600.
Cell culture and assessment of hTERT
Non-small lung cancer cells H1299 [13] were grown in RPMI 1640
media supplemented with 10% fetal bovine serum, 100 units/mL
penicillin and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 378C. Culture media were changed every
3 days. To establish stable cell lines in which the expression of
hTERT could be monitored by a reporter system, a 3.3 kbp DNA
fragment ranging from 3338 to þ1 bp of the hTERT gene was
subcloned upstream to a secreted alkaline phosphatase gene
(SEAP) and transfectectd into H1299 by electroporation. The
stable clones were selected using G418. The stable clones derived
from H1299 were cultured using conditions that are similar to
their parental cells.
Cytotoxicity assay
The tetrazolium reagent (MTT; 3-(4,5-di-methylthiazol)-2,5diphenyl tetrazolium bromide, USB) was designed to yield a
colored formazan upon metabolic reduction by viable cells
[14, 15]. Approximately 2 103 cells were plated onto each well
of a 96-well plate and incubated in 5% CO2 at 378C for 24 h. To
assess the in-vitro cytotoxicity, each compound was dissolved in
DMSO and prepared immediately before the experiments and
was diluted into the complete medium before addition to cell
cultures. Test compounds were then added to the culture
medium for designated various concentrations. After 48 h, an
amount of 25 mL of MTT was added to each well, and the samples
were incubated at 378C for 4 h. A 100-mL solution of lysis buffer
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Arch. Pharm. Chem. Life Sci. 2012, 345, 101–111
containing 20% SDS and 50% N,N-dimethylformamide was added
to each well and incubated at 378C for another 16 h. The absorbency at 550 nm was measured using an ELISA reader.
Telomere repeat amplification protocol (TRAP) assays
Telomerase activity was detected by a modified version of the
TRAP protocol [10, 16]. Telomerase products were resolved by
10% polyacrylamide gel electrophoresis and visualized by staining with SYBER Green. As a source of telomerase, the total cell
lysates derived from lung cancer cell line H1299 cells were used.
Protein concentration of the lysates was assayed using Bio-Rad
protein assay kit using BSA standards.
SEAP assay
Secreted alkaine phosphatase was used as the reporter system to
monitor the transcriptional activity of hTERT. Here, about 104
cells each were grown in 96-well plates and incubated at 378C for
24 h and changed with fresh media. Varying amounts of drugs
were added and cells were incubated for another 24 h. Culture
media were collected and heated at 658C for 10 min to inactivate
heat-labile phosphatases. An equal amount of SEAP buffer
(2 M diethanolamine, 1 mM MgCl2, and 20 mM L-homoarginine)
was added to the media and p-nitrophenyl phosphate was
added to a final concentration of 12 mM. Absorptions at
405 nm were taken, and the rate of absorption increase is determined [10, 17].
The present study was support by National Science Council Grants NSC 993112-B-010-001, 97-2311-B-010-005-MY3, 97-3112-B-010-013, 98-2113-M-016001 and 95-2113-M-016-002-MY2, respectively. The corresponding author
cordially dedicates this article to Prof. Dr. Dr. W. Wiegrebe (Regensburg/
Germany) on the occasion of his 80th birthday.
The authors have declared no conflict of interest.
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