In vivo evaluation of oral anti-tumoral effect of 3,4-dihydroquinazoline derivative on solid tumor

Article abstract of DOI:10.1016/j.bmcl.2011.11.083

An extension of our previously reported 3,4-dihydroquinazoline derivative is investigated. Oral anti-tumoral activity of 3,4-dihydroquinazoline derivative (KYS05090) as potent and selective T-type calcium channel blocker was in vivo evaluated against A549 xenograft in BALB/c^nu/nu nude mice. The rate of tumor volume increment in mouse model with KYS05090-treated group was remarkably slower than that of control group. With respect to tumor weight, it exhibited 60% and 67% tumor growth inhibition through oral administration of 1 and 5 mg/kg of bodyweight, respectively, compared to control and was more potent than paclitaxel (53%). In addition, KYS05090 (10 and 50 mg/kg, po) was found to have a marked analgesic effect in acetic acid-induced writhing test, whereas it did not show any effect on hot plate test.

Full text of DOI:10.1016/j.bmcl.2011.11.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1198–1201
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vivo evaluation of oral anti-tumoral effect of 3,4-dihydroquinazoline
derivative on solid tumor
Han Byul Kang a, , Hong-Kun Rim b,c, , Jin Yeong Park a, Heung Woo Choi a, Doo Li Choi a, Ji-Hyung Seo b,c
,
Kyung-Sook Chung b,c, Geun Huh a, Jungahn Kim a, Dong Joon Choo a, Kyung-Tae Lee b,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 Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
c Department of Biommedical Science, College of Medical Science, 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:
An extension of our previously reported 3,4-dihydroquinazoline derivative is investigated. Oral anti-
tumoral activity of 3,4-dihydroquinazoline derivative (KYS05090) as potent and selective T-type calcium
channel blocker was in vivo evaluated against A549 xenograft in BALB/cnu/nu nude mice. The rate of tumor
volume increment in mouse model with KYS05090-treated group was remarkably slower than that of
control group. With respect to tumor weight, it exhibited 60% and 67% tumor growth inhibition through
oral administration of 1 and 5 mg/kg of bodyweight, respectively, compared to control and was more
potent than paclitaxel (53%). In addition, KYS05090 (10 and 50 mg/kg, po) was found to have a marked
analgesic effect in acetic acid-induced writhing test, whereas it did not show any effect on hot plate test.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 October 2011
Revised 17 November 2011
Accepted 19 November 2011
Available online 28 November 2011
Keywords:
Oral anti-tumoral activity
A549 Xenograft
Solid tumor
Lung cancer
Analgesic effect
For several decades, medical oncologists have treated most of
their patients with intravenous (iv) anticancer drugs, and conse-
quently hospital services and clinical activities have been orga-
nized on the basis of this type of administration.1 However, in
the last six years the therapeutic scenario has been characterized
by a steady increase in the availability of oral anticancer drugs with
more than twenty oral anti-neoplastic drugs currently approved
for use in United States and Europe.2,3 Moreover, one-quarter of
all anticancer agents under development are oral drugs.4 The use
of an oral anticancer therapy affects many relevant aspects of the
clinical practice.
Calcium ion channels play a role in regulating calcium signaling
during cell proliferation.5 Among them, T-type calcium channel is
particularly the predominant influx mechanism in most solid can-
cers by which extracellular calcium enters.6 It was also found that
the T-type calcium channel blockers mibefradil and pimozide have
anti-proliferative effects on the mitogenic cell lines.7 Recently, we
reported that our 3,4-dihydroquinazoline derivative 1 (KYS05090:
Fig. 1) as a selective T-type calcium channel blocker exhibited 49%
tumor-weight inhibition compared with vehicle alone against
A549 xenograft in BALB/cnu/nu nude mice through intravenous
administration of 2 mg/kg of body weight (Table 1).8
Based on both the recent shift from iv to oral drug administra-
tion in oncology and the lower oral acute toxicity (LD50 = 693 mg/
kg) and the higher oral bioavailability (F = 98%) of KYS05090,8 we
decided to evaluate the oral anti-tumoral effect of KYS05090 on
solid tumor using A549 xenograft in BALB/cnu/nu nude mice in com-
parison with paclitaxel as positive control.9–11 A549 cells
(5 ꢀ 106 cells/mL) were injected subcutaneously (s.c.) into the left
flank region of BALB/cnu/nu nude mice and the tumors were allowed
to grow. When tumor nodules were clearly visible (ca. 3 mm3) after
8 days, KYS05090 with two doses, 1 and 5 mg/kg in co-solvent
(Ethanol: Cremophor EL: Distilled Water = 1:1:18) were orally
(po) administered five times per week for consecutive 39 days
and paclitaxel with 10 mg/kg dose in co-solvent was administered
through intraperitoneal (ip) injection twice per week during the
⇑
Corresponding author. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail address: ljy@khu.ac.kr (J.Y. Lee).
Both authors contributed equally to this work.
Figure 1. Structure of KYS05090.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.083

H. B. Kang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1198–1201
1199
Table 1
The pharmacological profiles of KYS05090
b
Calcium channel
blocking (IC50
Growth
Tumor weight
LD50
Fc
)
inhibition of inhibitiona
cancer cells
(GI50
)
T-type (a1G
0.041
)
N-type (a1B
)
A 549 (lung) A549 xenograft po
iv
40
l
M
4.9
lM
0.17 49% 693
l
M
98%
mg/kg mg/kg
a
Percentage of tumor-weight inhibition versus control at 2 mg/kg dose (once/
day).
b
Acute toxicity post single administration on ICR mice.
F denotes bioavailability.
c
same period. Tumor lengths and widths were measured weekly
using caliper and tumor volume was calculated as 1/
a
2 ꢀ length ꢀ width ꢀ height until animal sacrifice. The results are
documented in Figure 2 and Table 2. Figure 2a displays the effects
of KYS05090 on tumor growth in nude mice presented as tumor
volume over time. There was statistically significant dose-depen-
dent decrease in tumor volume with increasing administered dose.
The rates of tumor volume increment in mouse model with both
KYS05090-treated groups were remarkably slower than that of
control group (vehicle only). Specifically, KYS05090 at 1 mg/kg
dose showed the strong potency equal to paclitaxel as positive con-
trol and in particular KYS05090 at 5 mg/kg dose exhibited ca.
threefold efficacy than paclitaxel at 39th day as shown in Figure 2a.
At the end of experiment, all mice were weighed and sacrificed,
and their tumors were excised. Tumors were weighed and the
mean tumor weight was calculated. The tumor growth inhibition
rates (TGIR) were calculated as follows: Tumor growth inhibition
rates (TGIR,%) = 100 ꢀ (C ꢁ T)/C, where T is the average tumor
weight of the treated and C the average tumor weight of the con-
trol (vehicle only). The results presented in Figure 2b and Table 2
showed that KYS05090 exhibited better efficacy against A549
xenograft in BALB/cnu/nu nude mice than paclitaxel at both dosages:
60% and 67% inhibition at 1 and 5 mg/kg dose of KYS05090, respec-
tively, compared to 53% inhibition of paclitaxel. With respect to
body weight loss related with its chronic toxicity, there was no sta-
tistical difference between average total body weights in mice
treated with KSY05090, paclitaxel (positive control) and vehicle
(control). On the other hand, a little of body weight loss in mice
treated with 5 mg/kg dose of KSY05090 was observed during
19th–22nd day but the body weight was normally recovered after
then as shown in Figure 2c. The mortality rate in group treated
with KYS05090 was zero during the experimental period, which
supports the lower toxicity or the safety of KYS05090. This re-
vealed that KYS05090 may have a tumor suppressor function in
human lung cancer cells and could be a promising treatment in
oral anticancer therapy.
On the other hand, recent studies have indicated that T-type
calcium channels are present throughout the pain pathway and
may contribute to both peripheral and central sensitization.12,13
Additionally, some observations suggest that pharmacological
blockade of T-type channels may attenuate pain without signifi-
cant effects.14 Based on these studies, we decided to evaluate the
peripheral and central antinociceptive effects of KYS05090 be-
cause it is a potent T-type calcium channel blocker. The acetic
acid-induced writhing test and hot plate test is generally used
for the evaluation of peripheral and central antinociceptive activi-
ties, respectively.15,16 It has also been reported that acetic acid-in-
duced pain is related to peripheral nociceptive neurons17 and heat
induced pain is sensed by upper central nervous system.18 For the
evaluation of its peripheral antinociceptive effects, therefore,
KYS05090 with two doses (10 and 50 mg/kg) was orally adminis-
tered and after 1 h, mice were injected ip with a 0.7% (v/v) acetic
Figure 2. Oral anti-tumoral effect of KYS05090 in mouse xenografts models. A549
cells (5 ꢀ 106) were injected subcutaneously into the left flank of nude mice. Mice
were separated into four groups (n = 5 per each group). After 8 days when tumor
nodules were clearly visible, paclitaxel and KYS05090 were administered for
39 days. Control animals and the animals treated with paclitaxel or KYS05090 were
euthanized after 39 days and the tumors were excised and measured. Data points
represent a means SE; (a) tumor volumes in mice of each group; (b) tumor
weights in mice of each group measured on the last day of the experiment and the
photographs of representative tumors: (c) body weights in mice of each group. ⁄P
<0.05, ⁄⁄P <0.01, and ⁄⁄⁄P <0.001 compared with control group (Dunett’s test).
acid solution (10 mL/kg of body weight) as an irritant stimulus.
After a 5-min period, the number of writhes was recorded for
20 min. In the case of its central antinociceptive effect, KYS05090
with the same doses was orally administered and after 1 h, mice
were kept on the heated surface of the plate (55 0.5 °C) and the
time of latency was measured for each group at 0 and 30 min.

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H. B. Kang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1198–1201
Table 2
Oral anti-tumor efficacy of compound KYS05090 against A549 xenograft in nude micea
Compound
Dose (mg/kg)
ARb
No. of animals (n)
Tumor volumec (mm3)
Tumor weightc (g)
Controld
—
1
5
—
5
5
5
5
224.40 60.34
27.05 7.03
7.90 1.92
0.358 0.052
KYS05090d
po
po
ip
0.142 0.037 (60)e
0.120 0.022 (67)e
0.170 0.058 (53)e
Paclitaxeld
10
22.90 11.02
a
b
c
During 39 days after administration.
Administration route.
Data are expressed as mean S.E.
Co-solvent (Ethanol:Cremophor EL:DW = 1:1:18).
Tumor growth inhibition rates (%) = 100 ꢀ (C ꢁ T)/C.
d
e
Figure 3. Effects of KYS05090 in the acetic acid-induced writhing test in mice (n = 7 per each group) and hot plate test in mice (n = 10 per each group). (a) KYS05090 was
administered orally 1 h before injecting ip acetic acid (0.7% v/v, 10 mL/kg). (b) KYS05090 was administered orally 1 h before recording the hot plate latencies. The data show
the means S.D. ⁄⁄P <0.01 compared with the control group (Dunett’s test).
Scheme 1. Reagents and conditions: (a) biphenyl-4-carboxylic acid, DPPA, Et3N, toluene, rt to 100 °C, 72%; (b) PPh3Br2, Et3N, CH2Cl2, 0 °C, 77%; (c) toluene, rt, 85% for 6 and
77% for 9; (d) PhCH2NH2, TBD, 40 °C, ca. 89% for each reaction; (e) HCHO, H2, 10% Pd/C, CH3OH, 15 days (for 1.5 g of 7), rt, 70%; (f) 2 N HCl, EtOAc, rt, >99%; (g) (tBoc)2O, HCl,
MeOH, 0 °C to rt, 71% for 11 and 73% for 13; (h) LiAlH4, dry THF, 0 °C to reflux, 82% for 12 and 64% for 8.

H. B. Kang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1198–1201
1201
The effect of KYS05090 on acetic acid-induced writhing behavior
in mice is shown in Figure 3a: It dose-dependently and signifi-
cantly reduced the number of writhes induced by acetic acid
(0.7%, v/v, 10 mL/kg) in mice compared to control (vehicle) but
had a weaker effect than ibuprofen, a positive control. In the case
of hot-plate test, KYS05090 did not produce dose-independently
a reduction of the response to the hot-plate test compared to con-
trol as shown in Figure 3b. These overall results implicate that
KYS05090 as T-type calcium channel blocker has a peripheral
antinociceptive effect, not central antinociceptive effect.
In the course of scale-up synthesis of KYS05090 for its preclin-
ical trial, we met the unexpected synthetic problem as following:
The previously reported step (e) of Scheme 1, which is a one-pot
debenzylation and dimethylation of compound 7, took more than
2 weeks for the complete reaction of only 1.5 g-scale. This reaction
phenomenon may be due to a plausible catalyst poisoning of 3,4-
dihydroquinazoline moiety on the palladium catalyst as like the
deactivation role of quinoline on the Lindlar catalyst.19 In order
to avoid this longer and tedious step (e), therefore, we decided to
prepare directly N,N-dimethyl-N0-methylpentane-1,5-diamine (8)
from pentane-1,5-diamine (10) prior to the coupling reaction with
carbodiimide 4 as shown in Scheme 1. Diprotection of diamine 10
with di-tert-butyl dicarbonate using known procedure20 afforded
compound 11 in 71% yield. The reduction of both Boc groups with
lithium aluminum hydride gave compound 12 in 82% yield.21 The
repeated mono-protection and subsequent reduction of compound
12 afforded the target nucleophile 8 in 47% two-step yield. The
coupling reaction of 8 with carbodiimide 4 and the subsequent
reactions were successfully carried out to provide KYS05090 (1)
according to the previously reported procedure as shown in
Scheme 1.8
duction of KYS05090 has been finally optimized via the direct
preparation of side-chain present at C-2 position of 3,4-dihydro-
quinazoline ring. By applying this new scheme, ca. 100 g of
KYS05090 as a laboratory scale was prepared for further preclinical
trial.
Acknowledgment
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 (2011-
0027401).
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In conclusion, as a continuous part of our research for anti-tu-
moral agents for oral cancer therapy, in vivo efficacy of
KYS05090 as selective T-type calcium channel blocker was evalu-
ated against A549 xenograft in BALB/cnu/nu nude mice. KYS05090
exhibited not only potency in both patch-clamp and cellular as-
says, but also more potent oral anti-tumoral activity than paclit-
axel in this xenograft mice model, which is consistent with good
oral bioavailability. These overall results indicate that KYS05090
combined with a peripheral antinociceptive effect potentially
possess promising features as drug candidates for oral anticancer
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