Synthesis and biological evaluation of anilino substituted pyrimidine linked pyrrolobenzodiazepines as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.06.147

A series of new anilino substituted pyrimidine linked pyrrolo[2,1-c][1,4] benzodiazepine (PBD) conjugates were prepared and evaluated for their anticancer activity. The effects of four promising PBD conjugates on cell cycle of cancerous cell line A375 were investigated. These compounds showed the characteristic features of apoptosis like enhancement in the levels of p53, release of cytochrome c, and cleavage of PARP.

Full text of DOI:10.1016/j.bmcl.2010.06.147

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5232–5236
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of anilino substituted pyrimidine linked
pyrrolobenzodiazepines as potential anticancer agents
*
Ahmed Kamal , J. Surendranadha Reddy, M. Janaki Ramaiah, E. Vijaya Bharathi, D. Dastagiri,
*
M. Kashi Reddy, S. N. C. V. L. Pushpavalli, Manika Pal-Bhadra
Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2010
Revised 9 June 2010
Accepted 30 June 2010
Available online 29 July 2010
A series of new anilino substituted pyrimidine linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates
were prepared and evaluated for their anticancer activity. The effects of four promising PBD conjugates
on cell cycle of cancerous cell line A375 were investigated. These compounds showed the characteristic
features of apoptosis like enhancement in the levels of p53, release of cytochrome c, and cleavage of
PARP.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyrrolobenzodiazepine
DNA-binding affinity
Cytotoxicity
Cytochrome c
p53
Poly ADP ribose polymerase (PARP)
Apoptosis
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of
naturally occurring DNA-interactive antitumour antibiotics, exam-
ples of which include anthramycin, tomamycin, sibiromycin, chica-
mycin, prothracarin, DC-81 (3) and dextochrysin.1,2 The covalent
bond formation in the minor groove of DNA by nucleophilic attack
of 2-amino group of guanine base to form an amino linkage to C-11
is mainly responsible for the biological activities of PBDs.3,4 Re-
cently a series of C2-fluorinated PBDs have been synthesized and
evaluated for anticancer activity against a number of cancer cell
lines,5 in which fluorine substitution plays an important role for
their biological activity. In the past few years, several hybrid com-
pounds, in which known antitumour agents tethered to PBD moi-
ety, have been designed, synthesized and evaluated for their
biological activity.6,7 Moreover, we have been involved in the
development of new synthetic strategies8–10 for the preparation
of PBD ring system and also in the design of structurally modified
PBDs and their hybrids for the development of more potent anti-
cancer agents.11–14
Many chemotherapeutics are known to induce apoptosis, which
is a promising strategy for cancer drug discovery. Among the dif-
ferent approaches available to promote apoptosis, the develop-
ment of conjugates is one of the main strategies. From the
previous studies it is known that anilino primidines are reported
to possess apoptosis inducing ability,17,18 similarly some of the
PBD conjugates are also known to induce apoptosis.19,20 Therefore,
in continuation of our efforts in the development of apoptosis
inducers as potential anticancer agents, it has been considered of
interest to synthesize these PBD conjugates with a view to increase
the efficacy of the compounds, that is, additive or synergistic effect
in comparison to both the precursors. Therefore in continuation of
these efforts, we herein report the synthesis and biological evalua-
tion of some new hybrids wherein anilino substituted pyrimidine
moieties were linked to the PBD ring system.
Synthesis of pyrimidine intermediates 13a,b and 15 was carried
out from the compounds 11a,b which were prepared by the liter-
ature method.21 Compounds 11a,b were coupled to 5-bromo vale-
ryl chloride (12) to provide compounds 13a,b whereas this 11a
was treated with 4-(bromomethyl)benzoyl chloride (14) to obtain
compound 15 as shown in Scheme 1.
Synthesis of C8-linked anilino substituted pyrimidine—PBD
conjugates (4a–c and 5a,b) was carried out by employing the
hydroxy nitrothioacetal compounds 16a,b as the starting material,
which was prepared by previously reported methods.11–14 These
upon etherification with the intermediates 13a,b, and 15 using
K2CO3 in acetone provided the corresponding nitro thioacetals
An anilino substituted pyrimidine moiety present in the struc-
tures of agents like STI571 (Imatinib mesylate, gleevec (1)), nicoti-
nib (2) (Fig. 1) is considered responsible for the potent antitumor
properties.15,16
* Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.);
tel.: +91 40 27193236 (M.P.B.).
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in
(M. Pal-Bhadra).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.147

A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5232–5236
5233
CF3
O
H
N
N
N
N
N
H
H
N
H
N
N
N
N
N
N
2
N
N
O
N
HO
H
1
N
MeO
O
3
4a: R = 3-pyridyl; R1 = H
H
N
H
N
R
R
N
N
O
4b: R = 3,4,5-trimethoxyphenyl; R1 = F
H
H
4c: R = 3,4,5-trimethoxyphenyl; R1 = H
R1
N
O
O
N
N
MeO
O
N
O
5a: R = 3-pyridyl; R1 = H
5b: R = 3-pyridyl; R1 = F
R1
H
N
H
N
N
MeO
N
O
Figure 1. Chemical structures of biologically important imatinib mesylate or gleevec (1), nilotinib (2), DC-81 (3), and PBD conjugates (4a–c and 5a,b).
H
−
NH2
N
NH3+ NO3
NH
(i)
H
N
R
N
NO2
N
(iii)
NO2
6
NO2
7
10a,b
(iv)
+
O
O
(ii)
H
N
R
N
NH2
Br
R
R
N(CH3)2
8a,b
9a,b
a: R = 3-Pyridyl
N
b: R = 3,4,5-trimethoxyphenyl
11a,b
O
H
N
H
(v)
R
N
N
11a,b
11a
+
Br
Cl
12
N
O
13a: R = 3-Pyridyl
13b: R = 3,4,5-trimethoxyphenyl
Br
ClOC
H
N
H
N
R
N
(v)
+
Br
N
O
14
15: R = 3-Pyridyl
Scheme 1. Reagents and conditions: (i) H2NCN, HNO3, EtOH, reflux, 24 h, 58%; (ii) DMF–DMA, toluene, reflux, 24 h, 75%; (iii) NaOH, n-butanol, reflux, 48 h, 60%; (iv)
SnCl2ꢀ2H2O, HCl, rt, 2 h, 80%; (v) K2CO3, dry DMF, rt, 6 h, 85%.
(17a–c and 18a,b). Further, reduction of these nitro compounds by
using SnCl2ꢀ2H2O in MeOH followed by deprotective cyclization
employing HgCl2/CaCO3 afford the desired PBD conjugates (4a–c
and 5a,b) as shown in Scheme 2.22
DNA-binding ability of these PBD conjugates (4a–c and 5a,b)
was investigated by thermal denaturation studies using calf thy-
mus (CT) DNA. The increase in the helix melting temperature
pounds. Further, from the previous studies it was reported that,
PBD prefers G-rich sequences (50-G0GATCC-30) in DNA binding
which could be due to the affinity of PBDs in covalent interaction
with the free amino group attached to the N2 of guanine in the
DNA.23 Therefore, in order to investigate the binding capability of
these compounds to DNA at G-rich regions, restriction endonucle-
ase BamHI assay was performed. The pBR322 vector DNA was incu-
(D
Tm) for each compound was examined at 0 and 18 h incubation
bated with these compounds 4a,b and 5a,b at 4 and 8 lM
at 37 °C. Interestingly, all the compounds 4a–c and 5a,b elevated
concentrations and digested with BamHI enzyme. The results
the helix melting temperature of CT-DNA in the range of
showed that PBD conjugates could not inhibit DNA digestion at
1.2ꢁ7.1 °C. Compounds 5a,b have shown highest
D
Tm ranges from
4
l
M concentration. However, compounds 5a,b inhibited DNA
5.0 to 5.8 °C at 0 h and increased up to 6.8ꢁ7.1 °C after 18 h incu-
digestion at 8 M concentration as shown in Supplementary Figure
1. Further, it was observed from Figure 2 that one of the promising
l
bation. Moreover, the naturally occurring PBD, that is, DC-81 (3)
exhibits a
D
Tm of 0.7 °C under similar experimental conditions as
compounds 5b was found to inhibit the restriction digestion at
illustrated in Table 1.
8
lM concentration, whereas DC-81 showed similar effects at a
A quantitative restriction enzyme digestion (RED100) assay was
previously developed in which the inhibition of DNA cleavage by
BamHI is used to probe the DNA binding capability of PBD com-
higher concentration of 16 M. These results showed an enhanced
DNA binding ability for the compound 5b compared to the natu-
rally occurring DC-81(3).
l

5234
A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5232–5236
H
N
H
N
R
N
O
NO2
N
13a,b
CH(SEt)2
R1
N
O
MeO
17a−c
O
(ii), (iii)
H
H
N
R
N
N
N
O
H
N
O
N
MeO
HO
NO2
N
R1
CH(SEt)2
R1
4a: R = 3-pyridyl; R1 = H
O
4b: R = 3,4,5-trimethoxyphenyl; R1 = F
4c: R = 3,4,5-trimethoxyphenyl; R1 = H
(i)
MeO
O
16a: R1 = H
NO2
O
CH(SEt)2
R1
16b: R1 = F
H
H
N
R
N
N
N
15
MeO
18a,b
O
N
O
(ii), (iii)
N
O
H
H
H
R
N
N
N
N
MeO
R1
N
O
O
5a: R = 3-pyridyl; R1 = H
5b: R = 3-pyridyl; R1 = F
Scheme 2. Reagents and conditions: (i) K2CO3, dry CH3COCH3, 48 h, 80–89%; (ii) SnCl2.2H2O, CH3OH, reflux, 2 h; (iii) HgCl2, CaCO3, CH3CN–H2O, (4:1), 8 h, 52–59%.
Table 1
Thermal denaturation data for PBD conjugates 4a–c and 5a,b with calf thymus (CT)-
DNA
Compound
[PBD]/[DNA]
molar ratiob
D
Tm (°C)a after incubation at 37 °C for
0 h
18 h
4a
4b
4c
5a
5b
DC-81 (3)
1:5
1:5
1:5
1:5
1:5
1:5
2.0
1.5
1.2
5.0
5.8
0.3
2.1
2.5
1.4
6.8
7.1
0.7
a
For CT-DNA alone at pH 7.00 0.01,
separate determinations), all
Tm values are 0.1ꢁ0.2 °C.
For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100
and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM
sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
DTm = 68.5 °C 0.01 (mean value from 10
D
b
Figure 2. RED 100 assay: The figure in the top panel depicts the Red assay using
DC-81 (3) and figure in the bottom panel depicts the compound 5b. C, the Cut vector
DNA with BamHI enzyme in the absence of compound and UC, the uncut vector
DNA. The numbers 2, 4, 8, 16, and 32 depicts the concentration in micro molar of
the compound used in the RED assay.
lM
l
Compounds 4a–c and 5a,b were also evaluated for their cyto-
toxicity in selected human cancer cell lines of breast, oral, ovary,
colon, lung, prostate, and cervix by using sulforhodamine B (SRB)
cated that compound 5b to be the most effective among all the
compounds studied. Further, to confirm the synergistic effect of
the promising conjugates (5a,b), they were compared to both the
precursors 3 (DC-81) and 15 (leftside conjugate partner). As shown
in Supplementary Figure 4, both the conjugates (5a,b) exhibited an
increase in the cytotoxicity in relation to 3 (DC-81) and 15 in A375
cells.
In order to study the effect of these conjugates on cell cycle pro-
gression, human melanoma cell lines (A375) were treated with
compounds 4a,b, and 5a,b and analyzed using fluorescence acti-
vated cell sorter (FACS shown in Supplementary Fig. 5). Treatment
method. The compounds exhibiting GI50 610ꢁ5 M (10
lM) are con-
sidered to be active on the respective cell lines. Table 2 reveals that
these compounds exhibited broad-spectrum activity in all the cell
lines employed and the activity of the compounds ranged from
GI50 <0.1–2.7
trol that demonstrated GI50 values in the range of <0.1 to 2.1
whereas DC-81 showed GI50 values in the range of 0.11 to
2.37 M. Overall, significant cytotoxicity was observed for these
l
M. Adriamycin was employed as the positive con-
lM,
l
new PBD conjugates.
MTT assay was also carried out to identify the cytotoxic effect of
the four promising PBD conjugates (4a,b and 5a,b) on A375 cell
lines. IC50 values were found to be 5.5, 5.6, 1.1, and 0.16 lM for
4a,b and 5a,b, respectively. Further, the cell viability of A375 cells
was determined and the values found to be 72% (4a), 64.9% (4b),
of cells with these compounds at 4 lM concentration for 24 h in-
duced apoptotic effects up to 41.59%, 36.15%, 49.70%, and 83.86%
compared to 5.61% of control cells. Increased cells in sub G1 phase,
decrease of G1 and G2/M phase cells clearly showed that all the
compounds are effective in causing apoptosis as shown in Figure
3. Further, to evaluate the additive effect in the apoptosis inducing
ability, conjugate (5b) was compared to both the precursors 3
48% (5a), and 33.7% (5b), respectively, at 4
lM concentration as
shown in Supplementary Figures 2 and 3. The data clearly indi-

A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5232–5236
5235
Table 2
GI50 valuesa (in
lM) for compounds 4a–c and 5a,b in selected human cancer cell lines
Cell lines
4a
4b
4c
2.0 0.159
5a
5b
DC-81(3)
ADRi
MCF7b
A2780c
KBd
0.17 0.010
2.4 0.250
0.18 0.012
2.03 0.067
2.2 0.116
<0.1 0.007
2.5 0.089
2.3 0.086
0.16 0.008
0.17 0.007
2.3 0.016
0.16 0.007
0.18 0.011
0.17 0.002
0.17 0.012
1.8 0.069
0.15 0.003
0.18 0.008
2.1 0.038
0.12 0.001
2.7 0.186
2.5 0.073
0.14 0.007
0.13 0.009
0.14 0.003
0.15 0.005
0.17 0.006
<0.1 0.012
0.13 0.003
0.12 0.003
0.17 0.022
0.16 0.016
0.14 0.001
<0.1 0.008
0.13 0.003
0.12 0.006
0.14 0.004
0.12 0.005
0.13 0.008
0.14 0.003
<0.1 0.001
0.15 0.008
0.15 0.008
0.16 0.002
0.17 0.016
0.14 0.006
0.17 0.006
0.20 0.014
0.17 0.008
0.16 0.003
1.49 0.038
0.11 0.001
0.15 0.029
2.37 0.122
0.16 0.001
<0.1 0.012
<0.1 0.005
0.11 0.01
0.17 0.005
2.1 0.118
0.12 0.007
<0.1 0.006
<0.1 0.004
0.14 0.008
0.13 0.005
0.15 0.001
0.18 0.141
0.17 0.005
1.8 0.028
1.8 0.050
2.0 0.073
1.8 0.026
2.3 0.108
2.4 0.141
2.6 0.205
2.3 0.053
PC3e
SiHaf
GURAVd
DWDd
Colo205g
HOP62h
Zr-75-1b
A549h
a
50% growth inhibition and the values are mean of three determinations and are reported as mean standard error of the mean.
b
c
d
e
f
Breast cancer.
Ovarian cancer.
Oral cancer.
Prostate cancer.
Cervix cancer.
Colon cancer.
Lung cancer.
g
h
i
ADR (adriamycin).
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
G0
G1
S
Figure 4. Activation of tumor suppressor gene p53 and release of cytochrome c
from mitochondria. A375 cells were treated with concentration of PBD
conjugates for 24 h. C: control (untreated) C + D: control (untreated) + DMSO. b-
4 lM
G2/M
Actin was used as internal loading control.
C+D
4a
4b
5a
5b
Figure 5. PBD conjugates induced levels of cleaved PARP. A375 cells were treated
with 4 lM concentration of PBD conjugates for 24 h. b-Tubulin was used as loading
control. C: control (untreated) cells.
Figure 3. FACS analysis of cell cycle distribution of A375 cells after treatment with
PBD conjugates (4ab and 5ab) at 4 M for 24 h.
l
During apoptotic event, Procaspase-3 is degraded to active cas-
pase-3 and cleaves PARP.26 As we observed an increase in the lev-
els of cytochrome-c there could be an increase in the levels of
cleaved PARP. As expected the levels of cleaved PARP were more
prominent in the case of 5b as shown in Figure 5.
(DC-81) and 15 (leftside conjugate partner). As shown in Supple-
mentary Figure 6a and b, conjugate (5b) exhibited 99.58% of apop-
tosis in G0/G1 phase in comparison to 3 (86.73%) and 15 (68%) in
A375 cells.
Increased levels of p53 are found to be important in drug-in-
duced apoptosis.24 To investigate whether these PBD conjugates
are acting in p53 dependent apoptotic pathway, the expression
levels of p53 protein were checked by treating the cells with these
compounds and western blot analysis was carried out using p53
antibody. It is observed that p53 levels were up regulated in all
the compounds tested and it was more prominent in case of 4b
and 5b indicating a p53 dependent pathway as shown in Figure 4.
An important consequence of intrinsic apoptotic pathway is the
mitochondrial dysfunction and cytochrome c release. Cytosolic
cytochrome c is known to cause activation of caspase-3.25 To inves-
tigate the effect on cytochrome c A375 cells were treated with PBD
In the present study, a new series of anilino substituted pyrim-
idine linked pyrrolobenzodiazepine conjugates were prepared. The
thermal denaturation studies showed that these conjugates have
better DNA binding ability compared to DC-81. Moreover, these
binding studies were further validated by restriction endonuclease
BamHI digestion. Further, these PBD conjugates (4a–c and 5a,b)
showed good cytotoxicity against 11 human cancer cell lines. The
MTT proliferation assay for the four promising compounds in
A375 cells showed potent cytotoxicity at 4 lM. The FACS analysis
showed more population in sub-G1 phase indicating that all these
PBD conjugates have apoptosis inducing ability. It was observed
from the results that the p53 levels were enhanced. Pronounced in-
crease in the levels of cytosolic cytochrome c and cleaved PARP was
observed particularly, it was more pronounced in case of 5b.
Hence, these PBD conjugates induce apoptosis by acting through
the mitochondrial mediated pathway. Further, these results indi-
cated that the PBD conjugate (5b) is an effective cytotoxic agent
compared to DC-81 (3), with increased accumulation of cells in
conjugates at 4 lM concentration for 24 h and we observed that
there is an increase in the cytochrome c protein levels. However,
the enhancement in cytochrome c level was more pronounced in
case of compound 5b as shown in Figure 4. The data clearly shows
that PBD conjugates act through the involvement of mitochondria
in apoptotic pathway.

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5232–5236
13. Kamal, A.; Naseer, A. K.; Reddy, S.; Ahmed, S. K.; Kumar, M. S.; Juvekar, A.; Sen,
S.; Zingde, S. Bioorg. Med. Chem. Lett. 2007, 19, 5345.
14. Kamal, A.; Kumar, P. P.; Sreekanth, K.; Seshadri, B. N.; Ramulu, P. Bioorg. Med.
Chem. Lett. 2008, 16, 2594.
15. Deininger, M.; Buchdunger, E.; Druker, B. J. Blood 2005, 105, 2640.
16. Gorre, M. E.; Mohammed, M.; Ellwood, K.; Hsu, N.; Paquette, R.; Rao, P. N.;
Sawyers, C. L. Science 2001, 293, 876.
sub G1 phase of A375 cells. Further, it can be concluded that this
conjugate shows synergistic effect in comparison to the conjugate
partners. Finally, it suggests that one of the PBD conjugate like 5b
would be a potential lead for its development as a new anticancer
agent.
17. Sirisoma, N.; Kasibhatla, S.; Nguyen, B.; Pervin, A.; Wang, Y.; Claassen, G.;
Tseng, B.; Drewe, J.; Cai, S. X. Bioorg. Med. Chem. 2006, 14, 7761.
18. Sirisoma, N.; Pervin, A.; Nguyen, B.; Grundy, C. C.; Kasibhatla, S.; Tseng, B.;
Drewe, J.; Cai, S. X. Bioorg. Med. Chem. Lett. 2009, 19, 2305.
19. Wang, J. J.; Shen, Y. K.; Hu, W. P.; Hsieh, M. C.; Lin, F. L.; Hsu, M. K.; Hsu, M. H. J.
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Acknowledgment
The authors J.S.N.R., E.V.B., D.D.G., and M.K.R. are thankful to
CSIR, New Delhi, for the award of research fellowships.
20. Kamal, A.; Bharathi, E. V.; Ramaiah, M. J.; Dastagiri, D.; Reddy, J. S.; Viswanath,
A.; Sultana, F.; Pushpavalli, S. N. C. V. L.; Bhadra, M. P.; Srivastava, H. K.; Sastry,
G. N.; Juvekar, A.; Sen, S.; Zingde, S. Bioorg. Med. Chem. 2010, 18, 526.
21. Kil, K.-E.; Ding, Y.-S.; Lin, K.-S.; Alexoff, D.; Kim, S. W.; Shea, C.; Xu, Y.; Muench,
L.; Fowler, J. S. Nucl. Med. Biol. 2007, 34, 153.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.147.
22. Spectral data for compound (4a): Yield 59%; mp 114ꢁ116 °C; 1H NMR (CDCl3,
300 MHz): d 10.85 (s, 1H, NH), 9.27 (d, 1H J = 1.6 Hz), 8.93 (s, 1H, NH), 8.59 (d,
1H, J = 6.6 Hz), 8.48 (d, 1H, J = 5.2 Hz), 8.45ꢁ8.41 (m, 1H), 7.69 (d, 1H,
J = 3.6 Hz), 7.51ꢁ7.43 (m, 2H), 7.39 (s, 1H), 7.23 (d, 1H, J = 8.2 Hz), 7.17 (d,
1H, J = 4.4 Hz), 7.14 (d, 1H, J = 8.1 Hz,), 6.80 (s, 1H), 4.15 (t, 2H, J = 5.8 Hz), 3.90
(s, 3H), 3.94ꢁ3.54 (m, 3H), 2.59ꢁ2.49 (m, 2H), 2.32 (s, 3H), 2.04ꢁ1.90 (m, 4H),
1.31ꢁ1.24 (m, 4H); MS (ESI): m/z 606 (M+1)+; HRMS (ESI m/z) for C34H36N7O4,
calcd 606.2828, found 606.2819 (M+1)+. Compound (5a): Yield 54%; mp
108ꢁ110 °C; 1H NMR (DMSO, 300 MHz): d 10.18 (s, 1H, NH), 9.24 (d, 1H,
J = 1.8 Hz), 8.63 (dd, 1H, J = 3.6, 2.2 Hz), 8.51 (d, 1H, J = 5.2 Hz), 8.48 (d, 1H,
J = 8.1 Hz), 8.12 (s, 1H, NH), 7.85 (d, 2H, J = 7.4 Hz), 7.64 (d, 1H, J = 3.6 Hz),
7.56ꢁ7.36 (m, 6H), 7.19 (d, 1H, J = 8.2 Hz), 7.14 (d, 1H, J = 4.4 Hz), 6.80 (s, 1H),
5.25 (s, 2H), 3.96 (s, 3H), 3.89ꢁ3.47 (m, 3H), 2.34 (s, 3H), 2.13ꢁ1.97 (m, 2H),
1.82ꢁ1.56 (m, 2H); MS (ESI): m/z 640 (M+1)+; HRMS (ESI m/z) for C37H34N7O4,
calcd 640.2672, found 640.2668 (M+1)+. The detail spectral data of other
compounds are available in Supplementary data.
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