Anticancer activities and mechanisms of bisdioxopiperazine compounds probimane and MST-16.

Abstract

Bisdioxopiperazine compounds, including ICRF-154 and razoxane (ICRF-159, Raz), are anticancer agents developed in the UK specifically targeting tumor metastases. Further two bisdioxopiperazine derivatives, probimane (Pro) and MST-16, have been synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, PR China after 1980. Anticancer activities and mechanisms of Pro and MST-16 compared with Raz, especially for antiproliferative and antimetastatic effects in vivo and in vitro, have been systematically evaluated in this lab as well as by other authors in China. Novel molecular mechanisms especially relating to the inhibition of tumor metastasis between probimane and razoxane have been especially explored and explained, including pathways of inhibitions against calmodulin, sialic acid, lipoperoxidation, fibrinogen, cell-movement and the cell-cycle arrest. The distributions and excretions of (14)[C]-Pro in mice have been carefully monitored long before for explaining the relationship of pharmacological data between in vitro and in vivo evaluations. Pro is more soluble in water and more strongly active against tumors than Raz. In our point of view, Pro seems to inherit and retain most of the targets and pathways of other bisdioxopiperazine compounds currently in use and is cytotoxically more potent than the rest of bisdioxopiperazine compounds. Therefore, there is a great potential and significance for further investigations.

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78 Anti-Cancer Agents in Medicinal Chemistry, 2010, 10, 78-91
1871-5206/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Anticancer Activities and Mechanisms of Bisdioxopiperazine Compounds Probimane
and MST-16#
Da-Yong Lu1,* and Ting-Ren Lu2
1Shanghai University, Shanghai 200444, P.R. China; 2College of Sciences, Shanghai University, Shanghai 200444, P.R. China
Abstract: Bisdioxopiperazine compounds, including ICRF-154 and razoxane (ICRF-159, Raz), are anticancer agents developed in the
UK specifically targeting tumor metastases. Further two bisdioxopiperazine derivatives, probimane (Pro) and MST-16, have been synthe-
sized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, PR China after 1980.
Anticancer activities and mechanisms of Pro and MST-16 compared with Raz, especially for antiproliferative and antimetastatic effects
in vivo and in vitro, have been systematically evaluated in this lab as well as by other authors in China. Novel molecular mechanisms es-
pecially relating to the inhibition of tumor metastasis between probimane and razoxane have been especially explored and explained, in-
cluding pathways of inhibitions against calmodulin, sialic acid, lipoperoxidation, fibrinogen, cell-movement and the cell-cycle arrest. The
distributions and excretions of 14[C]-Pro in mice have been carefully monitored long before for explaining the relationship of pharmacol-
ogical data between in vitro and in vivo evaluations.
Pro is more soluble in water and more strongly active against tumors than Raz. In our point of view, Pro seems to inherit and retain most
of the targets and pathways of other bisdioxopiperazine compounds currently in use and is cytotoxically more potent than the rest of bis-
dioxopiperazine compounds. Therefore, there is a great potential and significance for further investigations.
Keywords: Bisdioxopiperazine, probimane, MST-16, razoxane, neoplasm metastasis, anticancer pharmacology, anti-neoplastic drugs, anti-
metastatic drugs, calmodulin, sialic acids, cell-cycle arrest, fibrinogen, cell-movement, lipoperoxidation, drug metabolism, lymphocyte-
rosette-formation.
BACKGROUNDS
Bisdioxopiperazine compounds (Biz), including ICRF-154,
razoxane (ICRF-159, Raz), ICRF-186 and ICRF-187 (two stereo-
isomers of Raz) and ICRF-193, developed in the UK, were among
the earliest agents found to be effective against a model of sponta-
neous metastasis (Lewis lung carcinoma) [1, 2]. Ever since that
time (1969), many studies have addressed their potential use and
mechanisms of action. Three main mechanisms of action have been
investigated before these investigations: potentiating the effect of
radiotherapy, overcoming multi-drug resistance (MDR) to daunoru-
bicin and doxorubicin in leukemia, and inhibiting topoisomerase II
and so forth [3-5]. More importantly, Raz has been licensed in
many countries as a cardioprotectant during anthrocycline treat-
ment. These kinds of researches have been large in volumes and
similarity in mechanism explanations, so we do not intend to review
them clearly in this paper. Since Biz compounds are unique agents
in their pharmacological action of metastases inhibition and are
conservative between compounds and somewhat undefined on the
important area of key targeting, especially in their anti-metastatic
mechanisms, further medicinal chemistry or pharmacological stud-
ies seems indispensable and should be stressfully focusing on
treatment-optimizing and related problems. Thereafter, probimane
[1,2-bis (N4-morpholine-3, 5-dioxopiperazine-1-yl) propane; AT-
2153, Pro] and MST-16 [1,2-bis (4-isobutoxycarbonyloxymethyl-3,
5-dioxopiperazin-1-yl) ethane] were synthesized at Shanghai Insti-
tute of Materia Medica, Chinese Academy of Sciences by Prof
Yun-Feng Ren and Prof Jun-Cao Cai [3-7]. The structural formulae
of the three bisdioxopiperazines are represented in Fig. (1). MST-
16, as a licensed drug in Japan since 1994, is allowed for directly
use in leukemia chemotherapy, mainly against adult T-cell leuke-
mia [8, 9]. As the principal researchers of Pro since the beginning
of my graduation study under the guidance of Prof Bin Xu and Prof
Jian Ding at Shanghai Institute of Materia Medica, Chinese Acad-
emy of Sciences from 1982 [10], [Lu, DY.; Anticancer activity of
three bisdioxopiperazines against lung cancer models. Master’s

*Address correspondence to this author at the Shanghai University, Shang-
hai 200444, P.R. China; E-mail: ludayong@sh163.net
#This work was supported by Shanghai Science and Technology Foundation
of High Education, 97A49
degree dissertation in Shanghai Institute of Materia Medica, Chi-
nese Academy of Sciences, March, 1986 (Chin); Lu, DY.; Antican-
cer effects and inhibitions on tumor cell movement of bisdi-
oxopiperazines (probimane and MST-16) in vitro. Doctorial degree
dissertation in Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, April 2005 (Chin)]. I have been systemati-
cally studying and pharmacologically comparing these three agents.
After my removing onto Shanghai University since 1987, I have
continued my study of Pro and further on changed focus a little bit
onto the molecular and mechanism study of the two compounds. In
this summary, most of the experiments over the past three decades
have been documented, tabulated, illustrated and discussed.
RESULTS
1. Antiproliferative Effects of Biz Compounds Against Experi-
mental and Human Tumor Lines In Vitro [4, 5]
The IC50 values of Pro against 10 the human tumor cell lines
originating from 7 organ types (Table 1) using a MTT assay have
been determined, and the IC50 values of Raz and MST-16 and Pro
for several of these tumor types have been compared and it has
been shown that Pro has greater anti-proliferative effects than Raz
or MST-16, especially against solid tumors (Tables 1-3). Also,
unlike most other anticancer drugs, the antiproliferative effects of
Pro and MST-16 steadily increased over 72 h (Fig. 2, Table 4),
longer retention of effectiveness of agents than most common anti-
cancer drugs. These data suggest that Pro can manifest the effec-
tiveness against tumor cells at lower dose ranges than Raz and re-
tain longer active interval than most of other anticancer drugs.
Antiproliferative effects of anticancer drugs against tumor cell
lines in vitro have been compared (Tables 2-4). Different anticancer
spectrum between Biz compounds and other anticancer drugs indi-
cates uniqueness of pharmacologic mechanisms and pathways of
Biz compounds.
2. Antitumor and Antimetastatic Effects on Lewis Lung Carci-
noma (3LL) In Vivo [3,5]
The antitumor and antimetastatic effects of Pro and Raz on LLC
have been shown in Tables 5 and 6. Pro and Raz at equitoxic doses
(LD5) showed a noticeable anticancer effect on primary tumor

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Anticancer Activities and Mechanisms of Bisdioxopiperazine Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 1 79
O
N
N
O
CH
CH3
CH2
NN
O
O
Razoxane
CH
CH3
CH2
NN
O
O
CH2
O
N
N
O
H2C
O
N
N
O
Probimane
O
N
N
O
CH2
CH2
N
N
O
O
H2C
O
O
O
CH2
O
O
O
MST-16

Fig. (1). Structural formulae of bisdioxopiperazine compounds.

Table 1. The IC50 Values of Pro in Different Tumor Cell Lines for 48 h. MTT Method was Used
Cell Origin Cell Types IC50 Values μ μM
Gastric SCG-7901 1.3672 ± 06230
MKN-28 24.314 ± 5.465
Colon HCT-116 14.476 ± 3.085
Mammary MDA-MB-435 45.325 ± 5.335
MDA-MB-468 22.169 ± 1.250
Pulmonary A549 0.02947 ± 0.02456
Leukemia HL-60 5.3417 ± 1.245
K562 4.786 ± 1.556
Uteric cervical HeLa 18.238 ± 1.112
Hepatic BEL-7402 42.457 ± 2.325

Table 2. Anti-Proliferative Effects of Anticancer Drugs Against Three Different Tumor Cell Lines In Vitro; Drug Exposure for 48 h
IC50 ?M
Compounds
P388 HL-60 HeLa
Doxorubicin 11.7 0.005 1.12
Vincristine No effect 0.05 4.56
5-fluorouracil 22.6 0.04 0.23
Probimane 64.6 1.97 5.12
ICRF-187 64.0 3.73 129
MST-16 5.23 33.4 26.4

Table 3. Dose-Response Relations Between Anti-Cancer Drugs for Anti-Proliferative Effect Against Human Leukemia Cell Line HL-60 for 24 h; *
P < 0.01; n = 3
Concentrations OD values Percentage Inhibition
Compounds
Mean ± SD %
Control -- 1.229 ± 0.125 --
Probimane 10.0 0.298 ± 0.010* 75.6
2.0 0.260 ± 0.005* 78.9
0.4 1.142 ± 0.010 7.1
0.08 1.199 ± 0.012 2.4
Doxorubicin 10.0 0.256 ± 0.021* 79.2

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80 Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 1 Lu and Lu
(Table 3) contd….
Concentrations OD Values Percentage Inhibition
Compounds
Mean ± SD %
2.0 0.266 ± 0.013* 78.3
0.4 0.312 ± 0.016* 74.5
0.08 0.408 ± 0.031* 66.9
5-Fluorouracil 5.0 0.421 ± 0.021* 65.6
1.0 0.518 ± 0.012* 57.9
0.2 0.585 ± 0.025* 54.4
0.04 0.892 ± 0.038* 27.5
Vincristine 5.0 0.425 ± 0.010* 65.4
1.0 0.423 ± 0.009* 65.6
0.2 0.401 ± 0.009* 67.4
0.04 0.394 ± 0.012* 68.0
* P<0.01.








Fig. (2). The IC50 values of probimane and MST-16 tested by exposure of
three human gastrointestinal tumor cell lines (SCG-7901, HCT-116 and
MDA-MB-468) for 48 h. MTT method was used.

growth (inhibition approximately 30-45%, P<0.05) and signifi-
cantly inhibited the formation of tumor metastases in the lung (in-
hibition of pulmonary metastasis, inhibitory rate>90%, P<0.001).
Primary LLC tumor growth was greatly inhibited more by Pro (48
%) than by Raz (40.3%) in a 20 day trial schedule, whereas the
inhibition by Pro (35.7%) was slightly less than that by Raz (40%)
in an 11 day trial schedule. It suggests the anticancer effect of Pro
seems to be more persistent than Raz in primary LLC tumor model.
The effects of Biz compounds on pulmonary metastasis of 3LL
were testified simultaneously by a bioassay: Table 7 showed that
Pro and Bim significantly inhibited the pulmonary metastasis of
3LL both following day 2 and day 8 injection schedules, but Raz
only significantly inhibited the pulmonary metastasis of 3LL fol-
lowing day 2 injection schedule. Pro inhibited the pulmonary me-
tastasis of 3LL more potently than Bim did at equitoxic dosage.
Comparatively, it seems that Pro has superior inhibition of pulmo-
nary metastasis of 3LL than Biz and Raz for its exclusively target-
ing potentiality [11].
3. Antitumor Effects Against Human Lung Adeno-Carcinoma
(LAX-83) In Vivo [3,5]
The anti-tumor activities of Pro and Raz on LAX-83 (Table
8)—a human pulmonary adeno-carcinoma tumor model trans-
planted in nude mice are significant. LAX-83 was found to be more
sensitive to Pro (80-100 mgKg-1 ip?5) with 55-60% inhibition
(P<0.01vs control), more than to Raz (40-60 mgKg-1, ip?5) with
25-32% inhibition (P<0.05 vs control). CTX, a known anticancer
drug (40 mgKg-1 ip?5), inhibited the growth of LAX-83 by 84%
(P<0.01vs control) the best one among all anticancer drugs tested in
the panel (Table not be listed here). Obvious necrosis in tumor tis-
sues was observed in histological evaluation of the CTX and Pro
treatment groups, but Pro produced larger vacuoles than CTX. CTX
was shown to be the most effective one in the LAX-83 model. The
anticancer effect of Pro against LAX-83 tumor growth was the
same as or even better than that of MTX, DDP or 5-Fu (Tables not
be listed here).
4. Inhibition and Blockage of Tumor Cell-Cycle Division [4]
To understand the mechanisms of action, the profile of antipro-
liferative and cytotoxicity characteristics against tumor growth of
Biz compounds has to be testified. The blockage chromosomal
segregation and G2/M phase arrests of Pro, Raz and MST-16 in
some human tumor cell lines in vitro were observed (Figs. 3-5).
These three compounds cause complete inhibition of tumor cell
division (80-95%) indicating linkages between blockage chromo-
somal segregation and G2/M phase arrests at the same dosage levels
(1- 4 μM). Similar dose-range between Pro, Raz and MST-16 sug-
gests that the two processes operate in the same course or cascade,
and are a universal pathway between Biz compounds; Antiprolif-
erative test (MTT) showed that Pro was more effective than MST-
16. Lacking parallels in effective ranges of Pro and MST-16 be-
Table 4. The Time-Response Relations Between Different Anticancer Drugs for Anti-Proliferative Effects Against Leukemia Cell Line HL-60;
N = 3, Probimane, Pro; 5-Fluorouracil, 5-Fu; Doxorubicin, Dox; Vincristine, VCR; ICRF-187, (+) Stereo-Isomer of Razoxane
Concentrations Percentage Inhibition %
Compounds
?M 24 h 48 h 72 h
Pro 10 75.6 78.5 75.9
5-Fu 2 65.8 53.5 51.1
Dox 4 78.3 72.4 72.3
VCR 2 65.4 59.0 57.1
ICRF-187 10 47.6 37.8 42.9
MST-16 10 47.8 5.6 0.0

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Anticancer Activities and Mechanisms of Bisdioxopiperazine Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 1 81
Table 5.
The Influence of Pro and Raz on Primary Tumor of LLC in C57/BL Strain Mice (Using Student’s t-test)
Compounds Dosage mg/kg/d Body Weight (g) Tumor Weight (g) Tumor Inhibition %
Control -- 23.3 / 24.4 2.80±0.04 --
Razoxane 20 23.3 / 23.4 1.61±0.03* 40.0
Probimane 30 23.4 / 21.6 1.91±0.03* 32.1
Probimane 60 23.3 /23.8 1.80±0.03* 35.7
Route: ip?7 daily. Duration of experiment was 11 days. * P<0.05 ( treatment vs vehicle control). The numbers of mice were 30 for the control group and 20 for each treatment group;
100 % survival was observed in each group.

Table 6.
The Influence of Pro and Raz on Primary and Metastatic Tumors of LLC in C57/BL Strain Mice
Compounds Dosage mg/kg/d Body Weight (g) PTI (%) MFCPM
Control --- 22.8 / 21.4 -- 30.9±7.3
Razoxane 20 22.7 / 21.5 40.3 1.2±0.5*
Probimane 30 23.3 / 22.5 42.0 1.5±0.5*
Probimane 60 23.3 / 20.3 48.0 1.0±0.2*
PTI (%) -- Primary tumor inhibition. MFCPM -- metastatic focus count per mouse. Route: ip?7 every 2 days. Duration of experiment was 20 days. * P<0.001 (treatment vs vehicle
control). The numbers of mice were 30 for both control group and each treatment group; 100 % survival was observed in each group.

Table 7. .
The Effects of Biz on Pulmonary Metastasis of Lewis Lung Carcinoma
Compounds Dosage ip?10 Initial Treated/d PTI% MNMCST
Control -- -- -- 9.5±0.6
Probimane 12 2 18.6 1.5±0.2**
Probimane 24 2 20.3 1.0±0.2**
Probimane 12 8 19.2 1.5±0.2**
Probimane 24 8 21.0 1.0±0.0**
Bimolane 6.5 2 18.3 3.5±0.2*
Bimolane 13 2 20.1 3.5±0.2*
Bimolane 6.5 8 13.1 4.0±0.4*
Bimolane 13 8 18.2 4.0±0.4*
Razoxane 6.5 2 19.2 1.5±0.2**
Razoxane 13 2 22.0 1.5±0.2**
Razoxane 6.5 8 15.0 9.0±0.0
Razoxane 13 8 15.8 8.5±0.8
Ten mice were used for each group, and each group was repeated once, *P<0.05, **P<0.01: PTI primary tumor inhibition; MNMCST, mean number of mice carrying secondary
tumors.

Table 8. Antitumor Activities of Pro and Raz on Human Tumor LAX-83 Using Subrenal Capsule Assay
Compounds Dosage mg/kg/d No mice Body weight (g) Tumor volume (mm3) Inhibition%
Control --- 16 19.2 / 21.0 39.8±3.2 --
Razoxane 40 12 20.8 / 21.5 29.7±3.0* 25
Razoxane 60 12 19.8 / 18.8 27.2±2.8* 32
Probimane 80 12 20.0 / 19.6 18.0±2.6** 55
Probimane 100 12 20.0 / 20.0 15.8±2.6** 60
CTX 40 12 21.0 / 20.9 6.4±2.0** 84
Route: ip?5 daily from the day after surgery. * P<0.05, ** P<0.001 (treatment vs vehicle control). Experiment was completed within 7 days. Tumor volume = 1/2?width2 ?length
(using t-test). CTX, cyclophosphamide.

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82 Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 1 Lu and Lu

















Fig. (3). G2/M phase arrests of human mammary tumor cell line (MDA-MB-435 cell) exposed to probimane at different concentrations for 20 h.























Fig. (4). G2/M phase arrests of human mammary tumor cell line (MDA-MB-435 cell) exposed to MST-16 and probimane for 20 h.

tween cytotoxicities (Pro 2-40 μM; MST-16 30-500 μM) and
chromosomal segregation—G2/M arrest (Pro, Raz and MST-16 1-
4μM) suggests that these two effects of Pro and MST-16 do not
follow the same inhibitory pathway. Some other anticancer molecu-
lar mechanisms presented following are proposed to be used for
explaining these differences between the three Biz compounds.
5. Inhibition on Calmodulin (CaM) Activity In Vitro
Our work shows that Pro (> 0.1mM) significantly inhibited
hemolysis by hypotonic and CaM activity in vitro, while Raz at
0.5mM almost showed no such activity (Tables 9, 10) [12-15]. It
suggests that Pro is more potent as an inhibitor of CaM than Raz, a
pathway of anticancer pharmacology. This action is parallel be-
tween their antihemolysis (Table 9) [12, 13] and inhibition of CaM
(Table 10) [14, 15] and antiproliferative effects against tumor mod-
els [4, 5]. Our early enzyme dynamic assay [the substrate concen-
tration (ATP)-response curve, dose (inhibitor, Pro)-response curve,
and time-response curve] showed that inhibitory rates of Pro are
unrelated to substrate (ATP) concentration, but related to CaM
concentrations (Fig. 6). It is possible that Pro is a CaM competitor
with CaM-like binding whose actions can be fulfilled by affecting
the reaction balance of substrate and antagonism on targeted en-
zymes of CaM such as Ca++-Mg++-ATPases [14, 15].
6. Inhibition of Lipoperoxidation in the Absence of ADR [16, 17]
The notification of antiradical activities (mitochondria swelling
and lipoperoxidation) by Biz compounds in the presence of anthro-
cycline (ADR and Dau) has been reported more than 1 decade by
Ms Zhang Y et al [18-20]. It has been used to explain the mecha-
nisms of cardioprotectant of anthrocycline—hypothesizing that Biz
compounds scavenge the free radicals activated and exaggerated by
anticancer anthrocyclines [18-20]. Therefore, there were some re-
ports stating these biological activities worked only in the presence
of anthrocycline [19]. Our work shows that Pro inhibited lipoper-
oxidation of rabbit and human red blood cells in vitro in the ab-
sence of anthrocycline in a dose-dependent manner (Tables 11, 12)
[16, 17]. The dose-range of Pro in inhibiting lipoperoxidation is
very similar with their antiproliferative effects against human can-