# Synthesis and Evaluation of Novel Organogermanium Sesquioxides As Antitumor Agents

Authors: Chun Li Zhang; Tai Hua Li; Shuang Huan Niu; Rong Fu Wang; Zhan Li Fu; Feng Qin Guo; Ming Yang
Journal: Bioinorganic Chemistry and Applications
 (2009)

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## Abstract

Five new organogermanium sesquioxides have been synthesized and characterized by elemental analysis and IR spectra. All the compounds were tested for antitumor activities against KB, HCT, and Bel cells in vitro. Compound 5 (γ-thiocarbamido propyl germanium sesquioxide) showed excellent antitumor activity, and its inhibition yield to KB, HCT, and Bel cells was 92.9%, 84.9%, and 70.9%, respectively. A rapid method was described for the labeling compound 5 with T99mc, and the optimum labeling conditions were investigated. The labeling yield is above 90% in pH 7.0, 20∘C, reaction time greater than 10 minutes, 1 mg of compound 5, and 0.075∼0.1 mg of SnCl2. The biodistribution of T99mc labeled compound 5 in nude mice bearing human colonic xenografts was studied. The result showed that the tumor uptakes were 0.73, 0.97, 0.87, and 0.62 ID%/g at 1-hour, 3-hour, 6-hour, and 20-hour postinjection, respectively. T/NT (the uptake ratio for per gram of tumor over normal tissues) was 18.3 for tumor versus brain and 5.81 for tumor versus muscle at 20-hour postinjection. The tumor clearance was slow. The results showed that compound 5 may be developed to be a suitable cancer therapeutic agent.


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## Body

## 1. Introduction

Germanium  is a constituent of many medicine plants such as ginseng root, and it is considered to play an important
role in the pharmacological effects of the plants [1]. It is reported that many organogermanium compounds can inhibit tumor and metastatic growth and modify immune response by inducing interferon-γ (IFN-γ), enhancing NK cell activity, and increasing peritoneal macrophage activity [2–5]. Besides, they are of extremely low toxicity [5, 6]. So, many researches focus on searching for effective and low-toxic antitumor drugs from organogermanium compounds [7–13].
Several types of organogermanium compounds have been investigated and found to possess antitumor effect [14–16], such as tetraalkylgermanium and alkylgermanium halide [17], spirogermanium [18], germanium sesquisulfide [19] or sesquioxide [20, 21], germatranes [22], Ge-porphyrinates, and germanium compounds containing phosphorus [23–25], of which Ge-132 (carboxyethylgermanium sesquioxide) has been clinically used [26, 27].For developing more effective antitumor agents from organogermanium sesquioxides, we synthesized five new organogermanium sesquioxides and investigated their antitumor activities against KB, HCT, and Bel cells. The compound with the highest antitumor activity was selected forT99mc labeling and biodistribution
study in nude mice bearing human colonic xenografts.


## 2. Experimental

### 2.1. Agents, Experimental Animals, and Instrumentation

All commercially available chemicals were of
analytical grade and used without further purification.T99mcO4− was provided by Syncor company with radiochemical purity greater than
98%.Experimental animals (Balb/c nude mice)
were provided by Experimental Animal Department of Chinese Academy of Medical
Sciences. The nude mice were implanted with human colonic carcinoma cells (LoVo cells). When the tumor size was about 1 cm, the mice were used for the
experiment.IR spectra were
recorded on a Bruker Equinox 55 spectrometer, US. Elemental analyses were
determined on a Yanaco CHN Corder MT-3 elemental analyzer. Mass spectrum (MS)
was detected on a Bruker APEX IV FTMS mass spectrometer. Radioactivity was
counted on a HY-901γ counter
provided by Sixin company.


### 2.2. Chemical Synthesis

The scheme of synthesis route is shown in Figure1. The
details of synthetic method were described by Niu [28].Figure 1
Scheme of synthesis route.


#### 2.2.1. Reagents

(1) Cl3GeH
Cl3GeH was prepared by the method reported by
Bai et al. [29]. 0.01 mol of GeO2, 2 mL of 50% H3PO2,
and 9 mL of HCl solution (11.9 M) were mixed and refluxed for  4 hours. A colorless
Cl3GeH solution was obtained. The solution was extracted three times
with ether to give Cl3GeH solution in ether.(2)β-Chloroformyl Ethyl Germanium Trichloride (I)
β-chloroformyl
ethyl germanium trichloride (I) was prepared by the method reported by Bai et al. [29]. 0.01 mol propenoic acid was added dropwise to 0.01 mol Cl3GeH
in HCl solution. A sticky precipitate was formed. After stirring for 4 hours, the
precipitate was filtered and recrystallized with CH2Cl2 to obtain 1.5 g (0.0055 mol) of I. 60% yield; mp: 70  ~  72°C.(3) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2, 3-Dicarboxylic Acid Anhydride (II)
7-oxabicyclo [2.2.1] hept-5-ene-2, 3-dicarboxylic acid
anhydride (II) was prepared by the method reported by Tian et al. [30].
0.5 mol of maleic anhydride, 0.6 mol of furan, and 50 mL of anhydrous
tetrahydrofuran were mixed and stirred to dissolve the maleic anhydride at 37°C. The solution was placed in room temperature for 2  ~  3 days until the
crystals were formed. The crystals were filtered, dried, and then
recrystallized with ethanol to obtain II. mp: 125  ~  127°C.(4) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (III)
7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine (III) was
prepared by the method reported by Liu et al. [31]. II was added to
25 mL of ammonia liquor (14.8 M) under stirring. The mixture was refluxed in
boiling water for 1 hour to yield yellow solution. The solution was cooled and
stood overnight. Needle crystals were formed. The crystals were filtered, dried,
and then recrystallized with 95% ethanol to obtain III. mp: 173  ~  175°C.(5) N-Amino-7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (IV)
N-amino-7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine
(IV) was prepared by the method reported by Liu et al. [31]. 5 mL of
50% hydrated hydrazine was added dropwise to 0.06 mol of I in water. The mixture
was refluxed in boiling water for 1 hour to yield purple solution and then
stood overnight. Lamellar crystals were formed. The crystals were filtered, dried,
and then recrystallized with distilled water to obtain IV. mp: 145  ~  146°C.(6) Ethyl Glutamate (V)
To prepare ethyl glutamate (V), a mixture of glutamic
acid (0.2 mol) and anhydrous ethanol (100 mL) was refluxed in an oil bath.
Dry hydrochloride gas was ventilated until the amino acid was dissolved
completely. Then we
stopped heating and continued to stir the reaction mixture for 1 hour.
Evaporation of the excess ethanol in vacuo resulted in light yellow oil. 40 mL
of distilled water was added, and the pH of the solution was increased by the
addition of 8 g of NaOH and stirred for 2 hours. The reaction mixture was
extracted three times with ethyl acetate (15 mL), dried (with anhydrous Na2SO4),
and concentrated in vacuo to give V as a light yellow oil.


#### 2.2.2. Synthesis of Germanium Sesquioxides of 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboxylic Acid Anhydride, and 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboximine Derivatives (Compound 1, 2, 3)

To a solution of II or III, IV (0.01 mol) in  an anhydrous solvent was added dropwise Cl3GeH (0.01 mol), and the solution was stirred overnight. The solvent was evaporated in vacuo, and the
residue was dissolved in distilled water (20 mL) and stirred for 2  ~  3 hours
to give a hydrolyzed product as a precipitate. The precipitate was filtered,
washed with aforementioned
solvent and distilled water, and dried to afford compound 1 or 2, 3.Compound 1:Elemental analysis: C: 36.21%, H: 2.62% (found); C: 36.42%,
H: 2.66% (calcd.).Compound 2:
Elemental analysis: C: 36.38%, H: 3.00%, N: 5.29% (found); C:
36.56%, H: 3.05%, N: 5.33% (calcd.).Compound 3:Elemental analysis: C: 34.63%, H:
3.29%, N: 10.01% (found); C: 34.58%, H: 3.24%, N: 10.09% (calcd.).


#### 2.2.3. Synthesis of Germanium Sesquioxide of Ethyl Glutamate (Compound 4)

To
a vigorously stirred solution of I in ether (10 mL) was added dropwise a
solution of V (4.0 g) in ether, and the solution was stirred for 6 hours. A
white sticky precipitate was formed and filtered off. The filtrate was
evaporated in vacuo to remove ether. Adding 10 mL of distilled water and
stirring for 6  ~  7 hours yielded a white precipitate. The precipitate was
filtered, washed three times with distilled water, and dried to give compound 4
as a white or light yellow powder.Compound 4:Elemental analysis: C: 41.02%, H: 5.64%, N: 4.01% (found); C: 40.61%, H: 5.64%, N: 3.95% (calcd.).


#### 2.2.4. Synthesis ofγ-Thiocarbamido Propyl Germanium Sesquioxide (Compound 5)

To a solution of propenyl thiocarbamide (0.01 mol) in anhydrous ethynol was added dropwise a
solution of Cl3GeH (0.01 mol) in ether, and the solution was stirred
for 3  ~  4 hours. The solvent was evaporated in vacuo to give an oily residue.
Addition of distilled water (20 mL) yielded a bright yellow precipitate. The
solution was kept stirring for 2 hours. The precipitate was filtered, washed
three times with distilled water, and dried to give compound 5.Compound 5:Elemental analysis: C: 21.96%, H: 4.23%, N: 13.09%
(found); C: 22.47%, H: 4.21%, N: 13.11% (calcd.).


### 2.3. Antitumor Activity

The antitumor activity in vitro was assayed by the MTT
method [32] using DMSO as solvent. The tumor inhibition yield and IC50 of the five organogermanium sesquioxides and Ge-132 against KB (human
nasopharyngeal cancer), HCT (human colonic cancer), and Bel (human liver
cancer)  cells were measured.


### 2.4. Radiolabeling with99mTc

Since compound 5 showed best antitumor activity, it was selected for further
radiolabeling and biodistribution studies. To the solution of compound 5 in NaOH solution (2 mol/L), 100μL freshly
prepared SnCl2·H2O solution was added, and pH was
adjusted to the required value. 3700  ~  5500 kBq
of T99mcO4− eluate was added, and the solution was stirred for 5 ~ 25 minutes to give the labeled compound. Labeling yield was measured by TLC
with polyamide membrane in the solvent of (CH3)2CHOH:NH4OH:H2O = 15:13:5 (v/v/v). Rf: labeled compound: 0.3 ~ 0.4, T99mcO4−:
0.5 ~ 0.6, T99mcO·xH2O: 0 ~ 0.1. Reaction conditions were
optimized by varying the concentration of germanium sesquioxide solution and
reducing agent (SnCl2), pH, temperature, and reaction time.


### 2.5. Biodistribution

Twelve nude mice bearing human colonic cancer
xenografts were injected in tail vein with 555 ~ 740 kBq ofT99mc
labeled compound 5. Three animals were sacrified at 1-hour, 3-hour, 6-hour, and
20-hour postinjection, respectively. Tissues and organs were excised, rinsed,
weighted, and counted in a γ counter. The uptake percentage of the injected
dose per gram of the measured organ (ID %/g) and the ratio of ID %/g of tumor
over normal tissues (T/NT) were calculated.


## 2.1. Agents, Experimental Animals, and Instrumentation

All commercially available chemicals were of
analytical grade and used without further purification.T99mcO4− was provided by Syncor company with radiochemical purity greater than
98%.Experimental animals (Balb/c nude mice)
were provided by Experimental Animal Department of Chinese Academy of Medical
Sciences. The nude mice were implanted with human colonic carcinoma cells (LoVo cells). When the tumor size was about 1 cm, the mice were used for the
experiment.IR spectra were
recorded on a Bruker Equinox 55 spectrometer, US. Elemental analyses were
determined on a Yanaco CHN Corder MT-3 elemental analyzer. Mass spectrum (MS)
was detected on a Bruker APEX IV FTMS mass spectrometer. Radioactivity was
counted on a HY-901γ counter
provided by Sixin company.


## 2.2. Chemical Synthesis

The scheme of synthesis route is shown in Figure1. The
details of synthetic method were described by Niu [28].Figure 1
Scheme of synthesis route.


### 2.2.1. Reagents

(1) Cl3GeH
Cl3GeH was prepared by the method reported by
Bai et al. [29]. 0.01 mol of GeO2, 2 mL of 50% H3PO2,
and 9 mL of HCl solution (11.9 M) were mixed and refluxed for  4 hours. A colorless
Cl3GeH solution was obtained. The solution was extracted three times
with ether to give Cl3GeH solution in ether.(2)β-Chloroformyl Ethyl Germanium Trichloride (I)
β-chloroformyl
ethyl germanium trichloride (I) was prepared by the method reported by Bai et al. [29]. 0.01 mol propenoic acid was added dropwise to 0.01 mol Cl3GeH
in HCl solution. A sticky precipitate was formed. After stirring for 4 hours, the
precipitate was filtered and recrystallized with CH2Cl2 to obtain 1.5 g (0.0055 mol) of I. 60% yield; mp: 70  ~  72°C.(3) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2, 3-Dicarboxylic Acid Anhydride (II)
7-oxabicyclo [2.2.1] hept-5-ene-2, 3-dicarboxylic acid
anhydride (II) was prepared by the method reported by Tian et al. [30].
0.5 mol of maleic anhydride, 0.6 mol of furan, and 50 mL of anhydrous
tetrahydrofuran were mixed and stirred to dissolve the maleic anhydride at 37°C. The solution was placed in room temperature for 2  ~  3 days until the
crystals were formed. The crystals were filtered, dried, and then
recrystallized with ethanol to obtain II. mp: 125  ~  127°C.(4) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (III)
7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine (III) was
prepared by the method reported by Liu et al. [31]. II was added to
25 mL of ammonia liquor (14.8 M) under stirring. The mixture was refluxed in
boiling water for 1 hour to yield yellow solution. The solution was cooled and
stood overnight. Needle crystals were formed. The crystals were filtered, dried,
and then recrystallized with 95% ethanol to obtain III. mp: 173  ~  175°C.(5) N-Amino-7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (IV)
N-amino-7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine
(IV) was prepared by the method reported by Liu et al. [31]. 5 mL of
50% hydrated hydrazine was added dropwise to 0.06 mol of I in water. The mixture
was refluxed in boiling water for 1 hour to yield purple solution and then
stood overnight. Lamellar crystals were formed. The crystals were filtered, dried,
and then recrystallized with distilled water to obtain IV. mp: 145  ~  146°C.(6) Ethyl Glutamate (V)
To prepare ethyl glutamate (V), a mixture of glutamic
acid (0.2 mol) and anhydrous ethanol (100 mL) was refluxed in an oil bath.
Dry hydrochloride gas was ventilated until the amino acid was dissolved
completely. Then we
stopped heating and continued to stir the reaction mixture for 1 hour.
Evaporation of the excess ethanol in vacuo resulted in light yellow oil. 40 mL
of distilled water was added, and the pH of the solution was increased by the
addition of 8 g of NaOH and stirred for 2 hours. The reaction mixture was
extracted three times with ethyl acetate (15 mL), dried (with anhydrous Na2SO4),
and concentrated in vacuo to give V as a light yellow oil.


### 2.2.2. Synthesis of Germanium Sesquioxides of 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboxylic Acid Anhydride, and 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboximine Derivatives (Compound 1, 2, 3)

To a solution of II or III, IV (0.01 mol) in  an anhydrous solvent was added dropwise Cl3GeH (0.01 mol), and the solution was stirred overnight. The solvent was evaporated in vacuo, and the
residue was dissolved in distilled water (20 mL) and stirred for 2  ~  3 hours
to give a hydrolyzed product as a precipitate. The precipitate was filtered,
washed with aforementioned
solvent and distilled water, and dried to afford compound 1 or 2, 3.Compound 1:Elemental analysis: C: 36.21%, H: 2.62% (found); C: 36.42%,
H: 2.66% (calcd.).Compound 2:
Elemental analysis: C: 36.38%, H: 3.00%, N: 5.29% (found); C:
36.56%, H: 3.05%, N: 5.33% (calcd.).Compound 3:Elemental analysis: C: 34.63%, H:
3.29%, N: 10.01% (found); C: 34.58%, H: 3.24%, N: 10.09% (calcd.).


### 2.2.3. Synthesis of Germanium Sesquioxide of Ethyl Glutamate (Compound 4)

To
a vigorously stirred solution of I in ether (10 mL) was added dropwise a
solution of V (4.0 g) in ether, and the solution was stirred for 6 hours. A
white sticky precipitate was formed and filtered off. The filtrate was
evaporated in vacuo to remove ether. Adding 10 mL of distilled water and
stirring for 6  ~  7 hours yielded a white precipitate. The precipitate was
filtered, washed three times with distilled water, and dried to give compound 4
as a white or light yellow powder.Compound 4:Elemental analysis: C: 41.02%, H: 5.64%, N: 4.01% (found); C: 40.61%, H: 5.64%, N: 3.95% (calcd.).


### 2.2.4. Synthesis ofγ-Thiocarbamido Propyl Germanium Sesquioxide (Compound 5)

To a solution of propenyl thiocarbamide (0.01 mol) in anhydrous ethynol was added dropwise a
solution of Cl3GeH (0.01 mol) in ether, and the solution was stirred
for 3  ~  4 hours. The solvent was evaporated in vacuo to give an oily residue.
Addition of distilled water (20 mL) yielded a bright yellow precipitate. The
solution was kept stirring for 2 hours. The precipitate was filtered, washed
three times with distilled water, and dried to give compound 5.Compound 5:Elemental analysis: C: 21.96%, H: 4.23%, N: 13.09%
(found); C: 22.47%, H: 4.21%, N: 13.11% (calcd.).


## 2.2.1. Reagents

(1) Cl3GeH
Cl3GeH was prepared by the method reported by
Bai et al. [29]. 0.01 mol of GeO2, 2 mL of 50% H3PO2,
and 9 mL of HCl solution (11.9 M) were mixed and refluxed for  4 hours. A colorless
Cl3GeH solution was obtained. The solution was extracted three times
with ether to give Cl3GeH solution in ether.(2)β-Chloroformyl Ethyl Germanium Trichloride (I)
β-chloroformyl
ethyl germanium trichloride (I) was prepared by the method reported by Bai et al. [29]. 0.01 mol propenoic acid was added dropwise to 0.01 mol Cl3GeH
in HCl solution. A sticky precipitate was formed. After stirring for 4 hours, the
precipitate was filtered and recrystallized with CH2Cl2 to obtain 1.5 g (0.0055 mol) of I. 60% yield; mp: 70  ~  72°C.(3) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2, 3-Dicarboxylic Acid Anhydride (II)
7-oxabicyclo [2.2.1] hept-5-ene-2, 3-dicarboxylic acid
anhydride (II) was prepared by the method reported by Tian et al. [30].
0.5 mol of maleic anhydride, 0.6 mol of furan, and 50 mL of anhydrous
tetrahydrofuran were mixed and stirred to dissolve the maleic anhydride at 37°C. The solution was placed in room temperature for 2  ~  3 days until the
crystals were formed. The crystals were filtered, dried, and then
recrystallized with ethanol to obtain II. mp: 125  ~  127°C.(4) 7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (III)
7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine (III) was
prepared by the method reported by Liu et al. [31]. II was added to
25 mL of ammonia liquor (14.8 M) under stirring. The mixture was refluxed in
boiling water for 1 hour to yield yellow solution. The solution was cooled and
stood overnight. Needle crystals were formed. The crystals were filtered, dried,
and then recrystallized with 95% ethanol to obtain III. mp: 173  ~  175°C.(5) N-Amino-7-Oxabicyclo [2.2.1] Hept-5-Ene-2,3-Dicarboximine (IV)
N-amino-7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximine
(IV) was prepared by the method reported by Liu et al. [31]. 5 mL of
50% hydrated hydrazine was added dropwise to 0.06 mol of I in water. The mixture
was refluxed in boiling water for 1 hour to yield purple solution and then
stood overnight. Lamellar crystals were formed. The crystals were filtered, dried,
and then recrystallized with distilled water to obtain IV. mp: 145  ~  146°C.(6) Ethyl Glutamate (V)
To prepare ethyl glutamate (V), a mixture of glutamic
acid (0.2 mol) and anhydrous ethanol (100 mL) was refluxed in an oil bath.
Dry hydrochloride gas was ventilated until the amino acid was dissolved
completely. Then we
stopped heating and continued to stir the reaction mixture for 1 hour.
Evaporation of the excess ethanol in vacuo resulted in light yellow oil. 40 mL
of distilled water was added, and the pH of the solution was increased by the
addition of 8 g of NaOH and stirred for 2 hours. The reaction mixture was
extracted three times with ethyl acetate (15 mL), dried (with anhydrous Na2SO4),
and concentrated in vacuo to give V as a light yellow oil.


## 2.2.2. Synthesis of Germanium Sesquioxides of 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboxylic Acid Anhydride, and 7-Oxabicyclo [2.2.1] Hept-2,3-Dicarboximine Derivatives (Compound 1, 2, 3)

To a solution of II or III, IV (0.01 mol) in  an anhydrous solvent was added dropwise Cl3GeH (0.01 mol), and the solution was stirred overnight. The solvent was evaporated in vacuo, and the
residue was dissolved in distilled water (20 mL) and stirred for 2  ~  3 hours
to give a hydrolyzed product as a precipitate. The precipitate was filtered,
washed with aforementioned
solvent and distilled water, and dried to afford compound 1 or 2, 3.Compound 1:Elemental analysis: C: 36.21%, H: 2.62% (found); C: 36.42%,
H: 2.66% (calcd.).Compound 2:
Elemental analysis: C: 36.38%, H: 3.00%, N: 5.29% (found); C:
36.56%, H: 3.05%, N: 5.33% (calcd.).Compound 3:Elemental analysis: C: 34.63%, H:
3.29%, N: 10.01% (found); C: 34.58%, H: 3.24%, N: 10.09% (calcd.).


## 2.2.3. Synthesis of Germanium Sesquioxide of Ethyl Glutamate (Compound 4)

To
a vigorously stirred solution of I in ether (10 mL) was added dropwise a
solution of V (4.0 g) in ether, and the solution was stirred for 6 hours. A
white sticky precipitate was formed and filtered off. The filtrate was
evaporated in vacuo to remove ether. Adding 10 mL of distilled water and
stirring for 6  ~  7 hours yielded a white precipitate. The precipitate was
filtered, washed three times with distilled water, and dried to give compound 4
as a white or light yellow powder.Compound 4:Elemental analysis: C: 41.02%, H: 5.64%, N: 4.01% (found); C: 40.61%, H: 5.64%, N: 3.95% (calcd.).


## 2.2.4. Synthesis ofγ-Thiocarbamido Propyl Germanium Sesquioxide (Compound 5)

To a solution of propenyl thiocarbamide (0.01 mol) in anhydrous ethynol was added dropwise a
solution of Cl3GeH (0.01 mol) in ether, and the solution was stirred
for 3  ~  4 hours. The solvent was evaporated in vacuo to give an oily residue.
Addition of distilled water (20 mL) yielded a bright yellow precipitate. The
solution was kept stirring for 2 hours. The precipitate was filtered, washed
three times with distilled water, and dried to give compound 5.Compound 5:Elemental analysis: C: 21.96%, H: 4.23%, N: 13.09%
(found); C: 22.47%, H: 4.21%, N: 13.11% (calcd.).


## 2.3. Antitumor Activity

The antitumor activity in vitro was assayed by the MTT
method [32] using DMSO as solvent. The tumor inhibition yield and IC50 of the five organogermanium sesquioxides and Ge-132 against KB (human
nasopharyngeal cancer), HCT (human colonic cancer), and Bel (human liver
cancer)  cells were measured.


## 2.4. Radiolabeling with99mTc

Since compound 5 showed best antitumor activity, it was selected for further
radiolabeling and biodistribution studies. To the solution of compound 5 in NaOH solution (2 mol/L), 100μL freshly
prepared SnCl2·H2O solution was added, and pH was
adjusted to the required value. 3700  ~  5500 kBq
of T99mcO4− eluate was added, and the solution was stirred for 5 ~ 25 minutes to give the labeled compound. Labeling yield was measured by TLC
with polyamide membrane in the solvent of (CH3)2CHOH:NH4OH:H2O = 15:13:5 (v/v/v). Rf: labeled compound: 0.3 ~ 0.4, T99mcO4−:
0.5 ~ 0.6, T99mcO·xH2O: 0 ~ 0.1. Reaction conditions were
optimized by varying the concentration of germanium sesquioxide solution and
reducing agent (SnCl2), pH, temperature, and reaction time.


## 2.5. Biodistribution

Twelve nude mice bearing human colonic cancer
xenografts were injected in tail vein with 555 ~ 740 kBq ofT99mc
labeled compound 5. Three animals were sacrified at 1-hour, 3-hour, 6-hour, and
20-hour postinjection, respectively. Tissues and organs were excised, rinsed,
weighted, and counted in a γ counter. The uptake percentage of the injected
dose per gram of the measured organ (ID %/g) and the ratio of ID %/g of tumor
over normal tissues (T/NT) were calculated.


## 3. Results

### 3.1. Preparations

The compounds were
prepared under mild condition. All the five compounds are powder and stable
under ordinary conditions. They all are not soluble in water, alcohol, slightly
soluble in DMSO, but compounds 3, 4, and 5 are easily soluble in 2 mol/L of
NaOH solution.The elemental analysis data and IR spectra were
consistent with the expected structure of the compounds. In IR spectra, the
wide absorption band at 800 ~ 900 cm-1 corresponds to the
characteristic region of νGe–O. The
weak absorption peak at about 550 cm-1 corresponds to the
characteristic region of νGe–C.MS was performed on compound 5 dissolved in the
mixture of DMSO and methanol. The solvent was selected because DMSO was the
solvent for measuring antitumor activity, and methanol was used to decrease the
viscosity of the solution to facilitate MS detection. MS showed the compound
containing two germanium atoms withm/z 456.815. The m/z value corresponds to
the molecular weight of the formula [Ge(CH2)3NHCSNHCH3]2O3 which may be formed by methylation of [Ge(CH2)3NHCSNH2]2O3 by methanol in DMSO.


### 3.2. Antitumor Activity

The antitumor activities of the five compounds and
Ge-132 at the concentration of 1, 10, and 50μg/mL were tested. All the
five compounds showed no antitumor activity at the concentration of 1 and 10 μg/mL. The
antitumor activities of the compounds at the concentration of 50 μg/mL are
listed in Table 1. The results of bioassay show that compounds 4, 5 exhibit
certain activities against cancer cells, but compounds 1, 2, 3 show no
antitumor activity. Compound 5 shows the
highest antitumor activity. Its inhibition yields to KB, HCT, and Bel cells are
92.9%, 84.9%, and 70.9%, respectively. IC50 is smaller than 10-7 M (IC50<10-7 M is considered as strong inhibition,
IC50 = 10-7 ~ 10-6 M as medium inhibition, and
IC50 = 10-6 ~ 10-5 M as weak inhibition). Ge-132
shows antitumor activity in vitro only to HCT cell, and the inhibition yield is
31.4%.Table 1
Inhibition yields (%) of the five compounds and Ge-132 at the concentration of 50μg/mL.
CellsCompound
1Compound
2Compound
3Compound
4Compound
5Ge-132KB000092.90HCT00041.584.931.4Bel000070.90


### 3.3.99mTc Labeling

Labeling yields of
compound 5 at different conditions are showed in Figure2. The compound shows
maximum labeling yield of above 90% at 1 mg of the germanium sesquioxide, pH 7.0 ~ 8.0,
20°C, with 0.075 ~ 0.1 mg of SnCl2 as reducing agent for
more than 10 minutes of reaction.Labeling yields (Lys) at different conditions.
(a)
Germanium sesquioxide: 0.1 mg, SnCl2: 0.1 mg,
T = 20°C, t = 10 minutes(b)
Germanium
sesquioxide: 0.1 mg, SnCl2: 0.1 mg, pH = 7.0, t = 10 minutes(c)
Germanium
sesquioxide: 0.1 mg, pH = 7.0,T = 20°C, t = 10 minutes(d)
pH = 7.0,
SnCl2: 0.1 mg, T = 20°C, t = 10 minutes(e)
Germanium
sesquioxide: 1 mg, SnCl2: 0.1 mg, T = 20°C, pH = 7.0


### 3.4. Biodistribution

The result of the biodistribution study of compound 5 in nude mice is tabulated in Table2. The uptakes of the compound in heart, brain, and muscle are low. It shows high uptake in liver, kidney, and lung. The
lung has the highest uptake at 3-hour and 6-hour postinjection.Table 2
Biodistribution
ofT99mc labeled compound 5 in nude mice bearing human
colonic cancer xenografts at various time intervals (% ID/g, n = 3, x¯±SD).
Tissues1 h3 h6 h20 hStomach10.10±3.895.57±0.891.27±0.250.59±0.18Small intestine1.29±0.581.06±0.113.09±3.270.33±0.14Kidney8.86±3.237.07±0.506.65±0.415.89±0.96Liver2.96±0.993.65±0.442.59±0.071.99±0.74Lung4.66±0.248.24±2.2412.40±4.944.56±3.28Muscle0.72±0.840.49±0.340.44±0.340.11±0.04Heart0.64±0.230.58±0.090.34±0.000.21±0.05Brain0.07±0.020.11±0.060.07±0.010.03±0.01Blood1.26±0.280.97±0.180.55±0.060.21±0.06Tumor0.73±0.100.97±0.200.87±0.040.62±0.23Tumor uptakes of compound 5 are 0.73, 0.97, 0.87, and 0.62
ID %/g at 1-hour, 3-hour, 6-hour, and 20-hour postinjection, respectively, which
are higher than those
of muscle, heart, and brain but lower than those of kidney, liver, and lung. The clearance of
the compound in tumor is relatively low (Figure3). At 20-hour postinjection,
the tumor uptake is higher than that of all other tissues except kidney, liver,
and lung. The maximum T/NT is the ratio of tumor to brain (18.30) and followed
by the ratio of tumor to muscle (5.81) at 20-hour postinjection. T/NT for tumor
versus blood increases with time with the maximum of 3.01 at 20-hour
postinjection (Table 3). The result of its relatively high tumor uptake and low
tumor clearance as well as its high tumor inhibition yield suggests that
compound 5 may be developed to be a suitable agent for cancer therapy.Table 3
T/NT
ofT99mc labeled compound 5 at various time intervals in nude mice bearing human colonic cancer xenografts (n = 3, x¯±SD).
Tissues1 h3 h6 h20 hStomach0.07±0.020.18±0.060.69±0.101.02±0.07Small intestine0.63±0.200.92±0.150.62±0.641.93±0.47Kidney0.09±0.040.14±0.020.13±0.020.10±0.02Liver0.26±0.060.27±0.080.34±0.030.31±0.02Lung0.16±0.020.12±0.010.04±0.010.27±0.28Muscle1.01±1.183.88±4.192.77±2.055.81±0.93Heart1.22±0.341.66±0.192.54±0.153.00±1.05Brain10.70±3.349.99±4.5312.90±2.9518.30±5.72Blood0.72±0.231.18±0.021.60±0.263.01±0.98Figure 3
ID %/g
of compound 5 in tumor, muscle, and blood at different time points.


## 3.1. Preparations

The compounds were
prepared under mild condition. All the five compounds are powder and stable
under ordinary conditions. They all are not soluble in water, alcohol, slightly
soluble in DMSO, but compounds 3, 4, and 5 are easily soluble in 2 mol/L of
NaOH solution.The elemental analysis data and IR spectra were
consistent with the expected structure of the compounds. In IR spectra, the
wide absorption band at 800 ~ 900 cm-1 corresponds to the
characteristic region of νGe–O. The
weak absorption peak at about 550 cm-1 corresponds to the
characteristic region of νGe–C.MS was performed on compound 5 dissolved in the
mixture of DMSO and methanol. The solvent was selected because DMSO was the
solvent for measuring antitumor activity, and methanol was used to decrease the
viscosity of the solution to facilitate MS detection. MS showed the compound
containing two germanium atoms withm/z 456.815. The m/z value corresponds to
the molecular weight of the formula [Ge(CH2)3NHCSNHCH3]2O3 which may be formed by methylation of [Ge(CH2)3NHCSNH2]2O3 by methanol in DMSO.


## 3.2. Antitumor Activity

The antitumor activities of the five compounds and
Ge-132 at the concentration of 1, 10, and 50μg/mL were tested. All the
five compounds showed no antitumor activity at the concentration of 1 and 10 μg/mL. The
antitumor activities of the compounds at the concentration of 50 μg/mL are
listed in Table 1. The results of bioassay show that compounds 4, 5 exhibit
certain activities against cancer cells, but compounds 1, 2, 3 show no
antitumor activity. Compound 5 shows the
highest antitumor activity. Its inhibition yields to KB, HCT, and Bel cells are
92.9%, 84.9%, and 70.9%, respectively. IC50 is smaller than 10-7 M (IC50<10-7 M is considered as strong inhibition,
IC50 = 10-7 ~ 10-6 M as medium inhibition, and
IC50 = 10-6 ~ 10-5 M as weak inhibition). Ge-132
shows antitumor activity in vitro only to HCT cell, and the inhibition yield is
31.4%.Table 1
Inhibition yields (%) of the five compounds and Ge-132 at the concentration of 50μg/mL.
CellsCompound
1Compound
2Compound
3Compound
4Compound
5Ge-132KB000092.90HCT00041.584.931.4Bel000070.90


## 3.3.99mTc Labeling

Labeling yields of
compound 5 at different conditions are showed in Figure2. The compound shows
maximum labeling yield of above 90% at 1 mg of the germanium sesquioxide, pH 7.0 ~ 8.0,
20°C, with 0.075 ~ 0.1 mg of SnCl2 as reducing agent for
more than 10 minutes of reaction.Labeling yields (Lys) at different conditions.
(a)
Germanium sesquioxide: 0.1 mg, SnCl2: 0.1 mg,
T = 20°C, t = 10 minutes(b)
Germanium
sesquioxide: 0.1 mg, SnCl2: 0.1 mg, pH = 7.0, t = 10 minutes(c)
Germanium
sesquioxide: 0.1 mg, pH = 7.0,T = 20°C, t = 10 minutes(d)
pH = 7.0,
SnCl2: 0.1 mg, T = 20°C, t = 10 minutes(e)
Germanium
sesquioxide: 1 mg, SnCl2: 0.1 mg, T = 20°C, pH = 7.0


## 3.4. Biodistribution

The result of the biodistribution study of compound 5 in nude mice is tabulated in Table2. The uptakes of the compound in heart, brain, and muscle are low. It shows high uptake in liver, kidney, and lung. The
lung has the highest uptake at 3-hour and 6-hour postinjection.Table 2
Biodistribution
ofT99mc labeled compound 5 in nude mice bearing human
colonic cancer xenografts at various time intervals (% ID/g, n = 3, x¯±SD).
Tissues1 h3 h6 h20 hStomach10.10±3.895.57±0.891.27±0.250.59±0.18Small intestine1.29±0.581.06±0.113.09±3.270.33±0.14Kidney8.86±3.237.07±0.506.65±0.415.89±0.96Liver2.96±0.993.65±0.442.59±0.071.99±0.74Lung4.66±0.248.24±2.2412.40±4.944.56±3.28Muscle0.72±0.840.49±0.340.44±0.340.11±0.04Heart0.64±0.230.58±0.090.34±0.000.21±0.05Brain0.07±0.020.11±0.060.07±0.010.03±0.01Blood1.26±0.280.97±0.180.55±0.060.21±0.06Tumor0.73±0.100.97±0.200.87±0.040.62±0.23Tumor uptakes of compound 5 are 0.73, 0.97, 0.87, and 0.62
ID %/g at 1-hour, 3-hour, 6-hour, and 20-hour postinjection, respectively, which
are higher than those
of muscle, heart, and brain but lower than those of kidney, liver, and lung. The clearance of
the compound in tumor is relatively low (Figure3). At 20-hour postinjection,
the tumor uptake is higher than that of all other tissues except kidney, liver,
and lung. The maximum T/NT is the ratio of tumor to brain (18.30) and followed
by the ratio of tumor to muscle (5.81) at 20-hour postinjection. T/NT for tumor
versus blood increases with time with the maximum of 3.01 at 20-hour
postinjection (Table 3). The result of its relatively high tumor uptake and low
tumor clearance as well as its high tumor inhibition yield suggests that
compound 5 may be developed to be a suitable agent for cancer therapy.Table 3
T/NT
ofT99mc labeled compound 5 at various time intervals in nude mice bearing human colonic cancer xenografts (n = 3, x¯±SD).
Tissues1 h3 h6 h20 hStomach0.07±0.020.18±0.060.69±0.101.02±0.07Small intestine0.63±0.200.92±0.150.62±0.641.93±0.47Kidney0.09±0.040.14±0.020.13±0.020.10±0.02Liver0.26±0.060.27±0.080.34±0.030.31±0.02Lung0.16±0.020.12±0.010.04±0.010.27±0.28Muscle1.01±1.183.88±4.192.77±2.055.81±0.93Heart1.22±0.341.66±0.192.54±0.153.00±1.05Brain10.70±3.349.99±4.5312.90±2.9518.30±5.72Blood0.72±0.231.18±0.021.60±0.263.01±0.98Figure 3
ID %/g
of compound 5 in tumor, muscle, and blood at different time points.


## 4. Discussion

Mironov et
al. synthesized  bis (2-carboxyethylgermanium) sesquioxide (now known as Ge-132)
in the 1960s [33]. In the 1970s, Japanese scholar K. Asai.
synthesized organogermanium pharmaceuticals with wide spectra of
pharmacological activities, in which he found Ge-132 showing antitumor activity [34].
Since then, a lot of studies showed that many organogermanium compounds can modify
immune function, inhibit tumor cell proliferation [35], induce tumor
cell apoptosis [36], and they are efficient in treating and
preventing cancers [37–40]. Besides, they can also relieve cancer-induced
pain [41], but the main problem of organogermanium compounds used as
antitumor agents is their low pharmacological effects, which makes them can
only be used as auxiliary agents in tumor therapy. So, development of more
effective antitumor organogermanium compounds is a valuable subject.In this
study, five new organogermanium compounds were synthesized and characterized by
elemental analysis data and IR spectra. The elemental analysis data and IR
spectra were consistent with the expected structure of the compounds. In IR
spectra, the wide absorption band at 800 ~ 900 cm-1 of the
characteristic region of νGe–O
provides support for Ge–O network structure. The weak absorption peak at about
550 cm-1 corresponds to the characteristic region of νGe–C. The
result shows that Ge–O network structure and Ge–C bond exist in the compounds.One of the
characteristics of germanium compounds is polymorphism [5]. The basic
formula of germanium sesquioxides is (GeR)2O3. So
germanium sesquioxides can be represented by [(GeR)2O3]n where R is the group conjugated with germanium atom and n is the degree
of polymerization. The degree of polymerization strongly influences the
toxicity of the compounds. It is reported that the value of “n” depends on the
way in which a solid product separates out of a solution of the compound.
Particle size and bulk density of the dried solid in batch-to-batch comparisons
are notoriously variable. Although there may be many polymorphic solid forms,
the aqueous solutions, once they are formed, are identical [5]. In
this study, the “n” value of compound 5 in the mixture of DMSO and methanol was
detected. The result showed that the compound existed in dimer form in the
solution.Two of the
five compounds showed certain antitumor activity at the concentration of 50μg/mL. Compound
5 showed very high antitumor activity with the inhibition yields of 92.9%,
84.9%, and 70.9% to KB, HCT, and Bel cells at the concentration of 50 μg/mL (0.117 mmol/L),
respectively. Compound 4 showed antitumor activity only to HCT cell with the
inhibition yield of 41.5%. The antitumor activities of compound 4 and compound
5 were higher than that of Ge-132 which has been clinically used. But Ge-132
showed its in vivo antitumor effect partly due to its inducing the antitumor
immunity of the host. Whether the new organogermanium compounds synthesized
have the in vivo immunopotentiating activity needs to be further investigated.Since many
organogermanium compounds have radiosensitive effect [36], it is
expected that radiolabeled organogermanium compounds may be more effective in
the therapy of cancer by synergic effect of cytotoxicity of the compounds and
radiation effect. We investigated the method of labeling compound 5 with T99mc.
The result showed that the compound can be successfully labeled with labeling efficiency
greater than 90%. The therapeutic radionuclide, 186Re or 188Re,
has similar chemical property to T99mc. So it is hopeful to label
the organogermanium compound with 186Re or 188Re for the
tumor therapy study. This work will be done later.Labeling
compound 5 withT99mc also allowed the biodistribution study of the
compound in nude mice bearing human colonic cancer. The biodistribution data showed
that the compound was mainly concentrated in kidney, lung, and next in liver.
Tumor uptake was higher than that of muscle, heart, and brain, but lower than
that of kidney, liver, and lung after 3-hour postinjection. The clearance of
the compound in tumor was relatively low. The result showed that the compound
has relative high tumor uptake and low tumor clearance, which facilitates
using the compound in cancer therapy.


## 5. Conclusion

A new class of organogermanium sesquioxides was
synthesized and characterized. The antitumor activity,T99mc labeling, and biodistribution were studied. Of the five compounds, compound 5 shows excellent antitumor activity, high tumor uptake, and slow clearance in tumor. It may be developed to be a suitable cancer therapeutic agent. The in vivo
antitumor effects of the organogermanium sesquioxides need to be further investigated.



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*Source: 908625-2009-06-02.xml*