Antitumor activity of 3,4-dihydroquinazoline dihydrochloride in A549 xenograft nude mice

Article abstract of DOI:10.1016/j.bmcl.2010.09.020

In the previous article we have reported that 3,4-dihydroquinazoline 1 is a potent and selective T-type calcium channel blocker that exhibited strong anti-cancer activity in vitro. Compound 1·2HCl was further in vivo evaluated against A549 xenograft in BALB/c nude mice, which exhibited 49% tumor-weight inhibition through intravenous administration of 2 mg/kg of body weight and was more potent than doxorubicin. Moreover, compound 1·2HCl has an oral bioavailability of 98% with LD₅₀ values of 693 mg/kg (po route) and 40.0 mg/kg (iv route) of body weight. In addition, its efficient scale-up synthetic method was developed.

Full text of DOI:10.1016/j.bmcl.2010.09.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6633–6636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antitumor activity of 3,4-dihydroquinazoline dihydrochloride
in A549 xenograft nude mice
Soo Yeon Jung a, So Hyung Lee a, Han Byul Kang a, Hang Ah Park a, Sun Ki Chang a, Jungahn Kim a,
Dong Joon Choo a, Chun Rim Oh b, Young Deuk Kim b, Ji Hyung Seo c, Kyung-Tae Lee c, Jae Yeol Lee a,
⇑
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b DongWoo Syntech Co., Ltd, Bondae-Ri Geumwang-Eup, Eumsung-Goon, Choongcheungbook-Do 369-901, Republic of Korea
c Kyung Hee East–West Pharmaceutical Research Institute and Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul 130-701,
Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2010
Revised 2 September 2010
Accepted 4 September 2010
Available online 21 September 2010
In the previous article we have reported that 3,4-dihydroquinazoline 1 is a potent and selective T-type cal-
cium channel blocker that exhibited strong anti-cancer activity in vitro. Compound 1Á2HCl was further
in vivo evaluated against A549 xenograft in BALB/c nude mice, which exhibited 49% tumor-weight inhibi-
tion through intravenous administration of 2 mg/kg of body weight and was more potent than doxorubicin.
Moreover, compound 1Á2HCl has an oral bioavailability of 98% with LD50 values of 693 mg/kg (po route) and
40.0 mg/kg (iv route) of body weight. In addition, its efficient scale-up synthetic method was developed.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anticancer activity
Pharmacokinetics
Acute toxicity
A549 Xenograft
Calciumis essential for livingorganisms, particularly in cell phys-
iology, where the movement of Ca2+ into and out of the cytoplasm
functions as a signal for many cellular processes including cell cycle
progression.1 Among various Ca2+ channels, T-type (low voltage
activated) Ca2+ channels have been known to play an important role
in controlling cell proliferation and differentiation in many tissues.2
In line with this molecular biology, the anti-cancer effects of a T-type
Ca2+ channel antagonists (or blockers) on tumor cells in vivo have
been reported by many researcher groups.3 Our group also have re-
ported the identification of 3,4-dihydroquinazoline compound 1,
which exhibits both selective/potent T-type calcium channel block-
ing effect and strong anti-cancer effect on cancer cell lines compara-
ble to doxorubicin in vitro.4 As a continuous work, the acute toxicity
study, A549 xenograft study and pharmacokinetic study for com-
pound 1 were performed. The results of these studies are reported
herein.
Before in vivo study, we had a problem with the previously
reported synthetic method for compound 1 which required multi-
step reactions, high expensive reagents and unstable intermediates.
Therefore, a more efficient synthetic procedure was desired to fur-
nish the quantities of compound 1 required for further in vivo bio-
logical evaluation study. We have modified the scale-up synthetic
procedure for compound 1 as shown in Scheme 1. In the previous
methods, iminophosphorane 3 was used as an intermediate for
key carbodiimide 5. However, this intermediate is too unstable to
be completely isolated and store for long time. Furthermore, 4-bi-
phenylyl isocyanate as a reagent for the reaction with compound 3
is highly expensive and also purification step via column chroma-
tography has to be avoided for scale-up preparation. As a solution
to these problems, we have developed an efficient two-step process
for carbodiimide 5: Curtius rearrangement using cheap reagent
biphenyl-4-carboxylic acid/diphenyl phosphorazidate (DPPA)/Et3N
for urea 4 and subsequent dehydration using PPh3Br2 and Et3N for
carbodiimide 5 as shown in Scheme 1 (path c and d).5 As a second
modified procedure, the reaction of ester compound 7 with benzyl-
amine and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as a catalyst
under solvent-free condition afforded directly amide 9 in higher
yield than the original two-step procedure (hydrolysis and amide
coupling reaction) as shown in Scheme 1 (path f).6 Finally, salt for-
mation to enhance aqueous solubility of compound 1 was carried
out using 2 N HCl in EtOAc to afford compound 1 dihydrochloride
salt, which became highly water soluble. The cytotoxic effect of both
free and salt forms on various cancer cell lines was then evaluated
using MTT assay for comparison.7 Both forms of compound 1
showed approximately similar cytotoxic activities against tested
cancer cells as shown in Table 1, which means that the salt formation
has no effect on cytotoxicity of compound 1 against cancer cells. This
modified procedure seems to be more efficient and economical than
the earlier reported method because of high overall yield (26%) and
⇑
Corresponding author. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail address: ljy@khu.ac.kr (J.Y. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.020

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S. Y. Jung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6633–6636
H2N
NH2
O
ref. 4
a
c
b
d
OCH3
O
PPh3
O
O
H3C
Bn
N
N
N
H
3
OCH3
6
OCH3
C N Ph-Ph
Bn
e
NH2
N
2
5
OCH3
NH
Ph-Ph
O
N
H
4
f
O
O
O
H3CO
HO
N
g
h
H
N
N
N
Bn
Bn
Bn
N
N
N
N
N
N
N
N
N
CH3
Bn
CH3
Bn
CH3
Bn
7
8
9
O
O
N
N
i
j
H
H
N
N
.2HCl
CH3
CH3
N
N
CH3
N
N
N
N
CH3
CH3
CH3
1
1 dihydrochloride
Scheme 1. Reagents and conditions: (a) PPh3, C2Cl6, Et3N, toluene, reflux, 60%; (b) 4-biphenylyl isocyanate, toluene, rt, 80%; (c) biphenyl-4-carboxylic acid, DPPA, Et3N,
toluene, rt to 100 °C, 72%; (d) PPh3Br2, Et3N, CH2Cl2, 0 °C, 77%; (e) toluene, rt, >99%; (f) PhCH2NH2, TBD, 40 °C, 89%; (g) LiOH, THF–H2O (1:1), 70 °C, >99%; (h) PhCH2NH2, HOBt,
EDC, THF–CH2Cl2 (1:1), 0 °C to rt, >79%; (i) HCHO, H2, 10% Pd/C, CH3OH, 4 days, rt, 70%; (j) 2 N HCl, EtOAc, rt, >99%.
Table 1
In vitro cytotoxic activity of 3,4-dihydroquinazolines 1 on cancer cell lines
Form of compound
Cancer cell lines (IC50 in lM)
A549 (lung)
HeLa (cervix)
LNCAP (prostate)
SKOV (ovary)
A172 (brain)
MDA-MB-231 (breast)
SKBR-3 (breast)
Free base
2HCl salt
5.17
5.00
4.04
3.11
4.70
5.19
5.00
5.22
4.83
4.61
2.65
1.64
2.17
1.83
fewer stages (seven steps). Therefore, we have prepared a large
quantity of compound 1Á2HCl using this modified procedure for its
following in vivo screening.
Compound 1Á2HCl was profiled for its acute toxicity to ICR male
mice using two kinds of administration route as shown in Table 2.8
In the case of oral administration, all the mice given a single oral
injection of 1Á2HCl at the dose of 250 mg/kg of body weight
showed no clinical sign and body weight loss and the mortality
rate was zero. At 500 mg/kg dose, however, clinical signs such as
diarrhea and soiled perineal region were observed on some of
tested mice on the injected day and disappeared on all of alive
mice after 3rd day post injection. The mortality rate was 2/5
Table 2
Acute toxicity of compound 1Á2HCl post single administration on ICR micea
Compound
Dose (mg/kg)
ARb
No. of animalsc
Sex
Clinical signs
Mortality
LD50
1Á2HCl
250
500
1000
26.6
34.6
45.0
—
po
po
po
iv
iv
iv
5
5
5
5
5
5
5/5
Male
Male
Male
Male
Male
Male
Male
Normal
0
2
4
0
1
4
0
693 mg/kg
Diarrhea; soiled perineal region
Diarrhea; soiled perineal region
Normal
Panting; inanimation; loss of locomotor activity; erosion
Panting; inanimation; loss of locomotor activity; erosion
Normal
40.0 mg/kg
—
Controld
po/iv
a
b
c
14 days post single administration.
Administration route.
Number of mice tested in the group.
10 mL/kg of saline, vehicle for compound 1Á2HCl salt.
d

S. Y. Jung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6633–6636
6635
Table 4
Pharmacokinetic parameters for compound 1Á2HCl in rat after iv administration
Parametera
Dose
5 mg/kg
10 mg/kg
AUC (ng h/mL)
Cmax (ng/mL)
t1/2 (min)
CL (mL/min/kg)
Vd (L/kg)
127.95 42.70
459.57 113.65
522.96 105.84
600.06 306.88
0.33 0.17
279.09 98.22
1026.37 475.85
1192.29 257.78
411.89 152.49
0.61 0.21
MRT (min)
F (%)
305.62 66.89
98.32 31.02
431.17 68.32
70.09 28.18
a
AUC is area-under-the-curve; Cmax is maximum concentration in plasma; t1/2 is
half-life in plasma; CL is plasma clearance; Vd is volume of distribution; MRT is
mean residence time; F denotes bioavailability.
mice presented as tumor volume over time. There was statistically
significant dose-dependent decrease in tumor volume with increas-
ing compound 1Á2HCl dose. In particular, compound 1Á2HCl at
2 mg/kg dose showed the strong potency equal to doxorubicin.
With respect to tumor weight, the results presented in Table 3
showed that compound 1Á2HCl through intravenous administration
of 1 mg/kg exhibited good efficacy against A549 xenograft in BALB/
c nude mice by 29% comparable to a doxorubicin (30%) at dosage
2 mg/kg. At 2 mg/kg dose, compound 1Á2HCl showed the 1.6-fold
potency (49%) than doxorubicin compared with vehicle alone. With
respect to body weight related with toxicity, there was no statistical
difference between average total body weights in mice treated with
compound 1Á2HCl and control, whereas significant body weight
loss in mice was continuously observed since 8th day after treating
with doxorubicin (data not shown). The present study implies that
compound 1Á2HCl could be effective in the treatment of human
lung cancer.
Based on these biological results, pharmacokinetic parameters
of compound 1Á2HCl were obtained from in sprague dawley rats
(n = 4) using two dosages (5 and 10 mg/kg) and summarized in Ta-
ble 4. Pharmacokinetic parameters generally showed the dose-
dependent pattern. First of all, compound 1Á2HCl is orally well ab-
sorbed at two dosages. The 98% oral bioavailability (F%) at low dose
(5 mg/kg) is particularly gratifying. The decreased oral bioavail-
ability at high dose (10 mg/kg) is thought to result from the satu-
ration at the absorption site. This pharmacokinetic profile shows
the possibility that compound 1Á2HCl would be developed as oral
anti-cancer agent.
Figure 1. Effects of compound 1Á2HCl on tumor growth in BALB/c nude mice
injected with 5 million human lung tumor A549 cells. The results are presented as
tumor volume over time.
(40%). At 1000 mg/kg dose, the mortality rate was 4/5 (80%) and
the same clinical signs were observed too. In addition, a little of
body weight loss was observed compared with the vehicle control
(p <0.05). Based on these data, oral LD50 value for compound
1Á2HCl was decided to be 693 mg/kg, which means that compound
1Á2HCl is relatively less toxic compared to other cancer chemother-
apeutic agents. In the case of intravenous administration, all the
mice given a single intravenous injection of 1Á2HCl at the dose of
26.6 mg/kg of body weight showed no clinical sign and body
weight loss and the mortality rate was zero. At the dose of
34.6 mg/kg, the mortality rate was 20% (1/5) and various clinical
signs such as panting, inanimation, loss of locomotor activity and
erosion were observed post injection and disappeared slowly on
alive mice. At high dose of 45.0 mg/kg, the same clinical signs were
observed and four of the treated mice were dead within 10 min
post injection. In particular, a little of body weight loss was ob-
served on the alive mouse compared with control, which may be
related with the toxicity of compound 1. Anyway, intravenous
LD50 value for compound 1Á2HCl was decided to be 40.0 mg/kg.
Based on these in vitro anti-cancer data and acute toxicity data,
we further evaluated the in vivo antitumor activity of compound
1Á2HCl against A549 xenograft in BALB/c nude mice model in com-
parison with doxorubicin as positive control and saline was used as
blank control.9,10 Compound 1Á2HCl with two doses (1 and 2 mg/
kg) and control with 5 mL/kg dose were administered once daily
through intravenous administration route for consecutive 28 days
and doxorubicin with 2 mg/kg was administered once per 2 days
through intravenous administration route for consecutive 4 weeks.
The results are documented in Figure 1 and Table 3. Figure 1 dis-
plays the effects of compound 1Á2HCl on tumor growth in nude
In conclusion, compound 1Á2HCl has relatively low toxicity,
in vivo good efficacy, good water solubility and excellent oral bio-
availability. All these results revealed that compound 1Á2HCl may
have a tumor suppressor function in human lung cancer cells and
could be a promising treatment in anti-cancer therapy. An efficient
and economical synthesis of large quantities of compound 1Á2HCl
has been developed.
Table 3
Antitumor efficacy of compound 1Á2HCl against A549 xenograft in nude micea
Compound
Dose (mg/kg)
ARb
No. of animals
Sex
Tumor weightc (g)
Tumor weight-inhibitiond (%)
Controle
1Á2HCl
—
1
2
iv
iv
iv
iv
4
4
4
4
Male
Male
Male
Male
0.537 0.172
0.381 0.055
0.273 0.078f
0.375 0.046
—
29
49
30
1Á2HCl
Doxorubicing
2
a
b
c
d
e
f
During 29 days post xenograft.
Administration route.
Data are expressed as mean S.E.
Percentage of tumor-weight inhibition versus control.
5 mL/kg saline, vehicle for compound 1.
p <0.05 versus the vehicle-treated control group.
10 mL/kg of doxorubicin hydrochloride in saline solution.
g

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S. Y. Jung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6633–6636
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This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2010-
0028197).
7. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
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