Thiourea derivatives, methods of their preparation and their use in neutron capture therapy of malignant melanoma

The present invention pertains to boron containing thiouracil derivatives, their method of preparations, and their use in the therapy of malignant melanoma using boron neutron capture therapy.

BRIEF DESCRIPTION OF THE INVENTION 
The present invention relates to new thiourea derivatives of the general 
formula 
##STR1## 
wherein: 
(A) R is hydrogen or an alkyl group containing from 1 to 8 carbon atoms, 
provided that where terminal double bonds originate from one of the two 
nitrogen atoms, R and/or the amide hydrogen is replaced; 
(B) X and X' are selected from the group consisting of 
##STR2## 
and &gt;C.dbd.Y, with Y.dbd.O or S; 
(C) m.dbd.0, 1 or 2 and o.dbd.0, 1 or 2; 
(D) 
##STR3## 
stands for a 1,2-dicarbaborane group, in which the 1- and 2-position of 
the carbon atoms correspond to A and B, and where n.dbd.0 or 1 but where 
not at the same time m, n, and o can all be zero, and where, if n.dbd.0, 
at least one of the groups R' and R" is a boron-containing group and the 
remaining groups R' and R" are hydrogen and/or an alkyl group with 1 to 8 
carbon atoms and their salts with physiologically compatible organic and 
inorganic bases or acids. 
The present invention also relates to methods for the preparation of the 
compounds of formula I, and their application in neutron capture therapy 
of malignant melanoma. 
BACKGROUND OF THE INVENTION 
Thioureas are known as substances which accumulate in malignant melanoma 
due to its active melanin synthesis. 
Malignant melanoma is a tumor of melanocytes. Its incidence is especially 
high among the fair-skinned population. In most industrial nations its 
incidence is increasing. 
Present treatment of melanoma consists of the surgical removal of the 
primary lesion. According to the histologically determined degree of 
classification, skin up to and including the regional lymph nodes are 
removed. Despite this, the five-year survival with melanomas grade II and 
higher, but also with melanomas grade I with high prognostic index (Kopf, 
Cancer, 59, 1236, 1987), is poor, as it apparently is not possible to 
remove all in-transit metastases of the skin and the lymph nodes. 
Radiation treatment of the afflicted limbs, in order to sterilize these 
metastases with or without radical surgery, has not yet been successful 
(Kynaston, Aust. N.Z. J. Surg., 48, 36, 1978). 
The known radiotherapeutic modality of neutron capture therapy utilizes the 
property of boron-10 (which represents 20% of the naturally occurring 
nuclide mixture and can be enriched from it) to capture a thermal neutron 
with high probability, as compared to the other nuclides of the body, and 
disintegrate upon capture to a helium-4 and a lithium-7 particle. Each of 
these particles is capable of sterilizing a cell with a single event 
(Gabel, Radiat. Res. 68, 307, 1984). 
Depending on the depth of the tumor and the energy spectrum of the neutron 
beam, at least 14 ppm boron-10 are necessary in the target tissue, for 
therapy to be successful, with a tumor-to-surrounding ratio of boron of 
around 10:1 (Fairchild and Bond, Int. J. Radiation Oncol. Biol. Phys., 11, 
831, 1985). For a thermal neutron beam and a tumor depth of 4 cm, tumor 
therapy is not possible with a boron ratio between tumor and surrounding 
tissue of 3:1 at any boron concentration; at a ratio of 10:1, 36 ppm boron 
are necessary. 
Neutron capture therapy (NCT) differs from other radiotherapy modalities 
inasfar as an external beam produces a high radiation dose only where a 
chemical compound has accumulated prior to irradiation. It differs from 
other chemotherapy modalities inasfar as the compound accumulated 
expresses its tumoricidal action only in the field of the beam. 
Coderre (Cancer Research, 48, 6313, 1988) has shown that 
p-dihydroxyborylphenylalanine (BPA) can accumulate physiologically in 
melanomas. Six hours after intraperitoneal injection, boron concentrations 
in the tumor of up to 30 ppm were found, with a tumor-to-blood and 
tumor-to-muscle ratio of around 5:1. After 24 hours, boron concentration 
in the tumor dropped and was too low for therapy. 
Mishima (Proc. 1st Int. Symp. NCT, 355, 1984) has reported the treatment of 
melanoma in swine with neutron irradiation following peritoneal injection 
of a total of 10 g BPA. 
Boronated thioureas, especially thiouracil derivatives, have been proposed 
for NCT by Fairchild (Cancer Res., 42, 5126, 1982). However, except for 
some attempted syntheses by Wilson (Australia-Japan Workshop on Neutron 
Capture Therapy for Malignant Melanoma, 1986) no boronated analogue has 
been described in the literature. The major difficulty in synthesizing 
such derivatives lies in the properties of the dihydroxylboryl group, 
which is easily cleaved off organic molecules by acids as well as alkali. 
The dihydroxyboryl group has been introduced into NCT by Schinazi and 
Prusoff (Tetrahedron Lett., 50, 4981, 1978; J. Org. Chem., 50, 841, 1985). 
The aim of the present invention is to provide stable boron-containing 
thiourea derivatives for neutron capture therapy, and to give procedures 
for their syntheses. 
DETAILED DESCRIPTION OF THE INVENTION 
Within the scope of this invention lie all cyclic thiourea derivatives, in 
which the cyclic portion, aside from the grouping 
##STR4## 
wherein R is H or lower alkyl consists of carbon, nitrogen, oxygen, sulfur 
or combinations of these elements, wherein the cyclic portion can be 
saturated or unsaturated, and the number of the links connecting the two 
nitrogen atoms of the thiourea fragment is 2 to 10, in which the carbon 
and/or nitrogen atoms of the cyclic part carry hydrogen atoms or where the 
hydrogen atoms are replaced by alkyl groups with 1 to 8 carbon atoms, and 
in which the cyclic part contains at least one boron-containing group. The 
boron containing group can be connected via a single or several bonds to 
different atoms of the cyclic part. 
Representative compounds of the present invention include: 
##STR5## 
The following are the complete chemical names for compounds A-Q depicted 
above: 
(A) 5-dihydroxyboryl-2-thiouracil 
(B) 5-dihydroxyboryl-2,4-dithiouracil 
(C) 5-dihydroxyboryl-6-propyl-2-thiouracil 
(D) 5-dihydroxyboryl-6-propyl-2,4-dithiouracil 
(E) 4-(3-carboranylpropyl)-thiyl-pyrimidine-2-thiol 
(F) 4-(3-nidocarboranylpropyl)-thiyl-pyrimidine-2-thiol 
(G) 5-(N-(3-carboranylpropyl)-N,N-dimethyl)amino-2-thiouracil 
(H) 4-dihydroxyboryl-1-methylimidazole-2-thiol 
(I) 5-dihydroxyboryl-1-methylimidazole-2-thiol 
(K) 4-dihydroxyboryl-5-methyl-1-methylimidazol-2-thiol 
(L) 5-dihydroxyboryl-4-methyl-1-methylilizadol-2-thiol 
(M) 4-undecahydrododecaboranylthiyl-pyrimidin-2-thiol 
(N) 4-undecahydrododecaboranylamino-pyrimidin-2-thiol 
(O) 5-carboranylmethyl-1-methyl-2-thio-1,3,4-triazol 
(P) 5-nidocarboranylmethyl-1-methyl-2-thio-1,3,4-triazol 
(Q) N,N'-thiocarbonyl-1,2-bis(aminomethyl)nidocarboran. 
Of the compounds of the present invention depicted by Formula I above, the 
most preferred materials are: 
5-dihydroxyboryl-2-thiouracil 
5-dihydroxyboryl-2,4-dithiouracil 
5-dihydroxyborylboryl-6-propyl-2-thiouracil 
5-dihydroxyboryl-2,4-dithiouracil 
5-dihydroxyboryl-1-methylimidazole-2-thiol and 
4-dihydroxyboryl-1-methylimidazole-2-thiol. 
Cyclic thioureas according to the present invention can be obtained by: 
reacting, with boron compounds, a pre-formed cyclic thiourea derivative 
with suitable reactive groups, where the thiourea moiety of the end 
product is present as iso-thiourea; by reacting an open-chain thiourea 
moiety with a suitable boron-containing compound, followed by cyclization 
to the desired end product; or by reacting a suitable boron compound with 
a compound such that the thiourea moiety is introduced during the 
formation of the cyclic structure. 
More specifically, thiourea derivatives of the general formula 
##STR6## 
wherein R, X, X', m and o are as defined above are prepared when a 
compound of the general formula 
##STR7## 
wherein R, X, X', m and o are as defined above in which the group(s) R' 
and R", which are the boron-containing groups in the final product, are 
the same as in the final product, or are present as dialkyloxy boryl 
groups, preferentially diothanolaminoboryl groups, is reacted with a Lewis 
acid and the dialkoxy groups, when present, are transformed hydrolytically 
to dihydroxyl boryl groups. 
In the preparation of iso-thiourea derivatives exemplified by formula III 
above, where at least one of the groups R' and R" are -E-(alkylene)-z, 
with --E--=--O--, --S--, &gt;NR'", and where R'" is hydrogen or alkyl with 
C.sub.1 to C.sub.8, and where the alkylene group contains 1 to 8 carbon 
atoms, and where Z is the 1,2-dicarba-closo-dodecaboranyl or 
1,2-dicarba-nido-undecaboranyl group, preferably a compound of the general 
formula III, in which the position(s) that bear(s) the -E-(alkylene)-Z 
group in the final product, is(are) present as --OH, --SH, or &gt;NR'"H, are 
reacted with the corresponding 
1-(omega-haloalkyl)-1,2-dicarba-closo-dodecaborane 
##STR8## 
with halo=Cl, Br, I and q=1 to 8, under neutral or basic conditions. 
The preferred Lewis acids for use in the preparation of the compounds of 
formula II above include AlBr.sub.3, AlCl.sub.3, and BBr.sub.3. 
Thiourea derivatives of the general formula 
##STR9## 
wherein E=--O--, --S--, &gt;NR'", with R'"=H or alkyl with 1 to 8 carbon 
atoms and p=1 to 8, and Z is the 1,2-dicarba-closo-dodecaboranyl group, 
are prepared when a compound of the formula 
##STR10## 
wherein E is as defined above is reacted with a 
1-(omega-haloalkyl)-1,2-dicarbacloso-dodecaborane of the formula 
##STR11## 
wherein the halogen is Cl, Br or I and p=1 to 8 under neutral or basic 
conditions. 
Thiourea derivatives of the formula 
##STR12## 
wherein R is as defined above and r=1 to 5 are prepared when a 
thiosoimicarbazide of the formula 
##STR13## 
wherein R is as defined above is reacted with an omega-carboranyl acyl 
halide of the formula 
##STR14## 
wherein halo and r are as defined above and the reaction product is 
cyclized by action of a base. 
Boron distribution in tissue was measured with 
5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), and 
5-dihydroxyboryl-2-thiouracil (BTU) using quantitative neutron capture 
radiography (Gabel, Cancer Res., 47, 5451, 1987). BTU and BPTU exhibited 
several advantages compared to the compound p-boronophenylalanine, so far 
best suited for accumulation of boron in melanoma: (a) BTU and BPTU 
accumulate in tumors for long time periods (days to weeks). For treatment 
of tumor it is advantageous if the accumulation once obtained can be 
retained, as several subsequent administrations of the compound will lead 
to higher accumulation. In addition, those cells can be loaded with boron 
that were, during the first administration, in a phase of their cell cycle 
not optimal for boron uptake. Also, the time between administration and 
irradiation can be changed in larger margins. A long period of 
accumulation is essential for a protracted or a fractionated irradiation; 
(b) BTU and BPTU leave the other non-melanoma organs of the body rapidly. 
The compounds leave the body via kidney and gall bladder. Half times of 
BTU in blood and muscle are around 2 hours, for BPTU around 6 hours. 
Irradiation can thus be initiated shortly after the last administration; 
(c) The compounds of this invention achieve boron concentration ratios 
between the tumor and its direct surrounding (blood, muscle, skin) of up 
to 50:1; and (d) Maximum concentrations in the tumor of 100 ppm boron and 
more can be achieved. 
Tables 1-4 show the distribution of BTU and BPTU in various melanomas. 
TABLE 1 
______________________________________ 
Distribution of 5-Dihydroxyboryl-2-thiouracil 
(Compound A) in Harding-Passey Melanoma 
Dose Time Tumor Ratio Tumor To: 
(mg/kg) 
(hr) (ppm) Blood Muscle 
Brain 
______________________________________ 
300 3 40-80 &gt;5 &gt;5 &gt;8 
______________________________________ 
TABLE 2 
______________________________________ 
Distribution of 5-Dihydroxyboryl-6-propyl-2-thiouracil 
(Compound C) in B16 Melanoma 
Dose Time Tumor Ratio Tumor To: 
(mg/kg) 
(hr) (ppm) Blood Muscle 
Brain 
______________________________________ 
300 12 3-10 &gt;10 approx. 
approx. 
30 20 
______________________________________ 
TABLE 3 
______________________________________ 
Distribution of 5-Dihydroxyboryl-2-thiouracil (Compound A) 
in Balb/cI Mice Carrying Harding-Passey Melanoma 
Dose Time Tumor Ratio Tumor To: 
(mg/kg) (hr) (ppm) Blood Muscle Brain 
______________________________________ 
300 (t = 0) 
240 (t = 3) 
12 80-100 &gt;5 &gt;8 &gt;15 
______________________________________ 
TABLE 4 
______________________________________ 
Distribution of 5-Dihydroxyboryl-6-propyl-2-thiouracil 
(Compound C) in C57bl Mice with B16 Melanoma 
Dose Time Tumor Ratio Tumor To: 
(mg/kg) (hr) (ppm) Blood Muscle Brain 
______________________________________ 
190 4 4-90 10 5 4 
(mean 41) 
______________________________________ 
The following examples are to illustrate the invention, especially 
concerning the procedures according to this invention, and the use of the 
resulting products. In this "carborane" designates the 1,2-dicarba, 
closo-dodecarborane group, "nidocarborane" the 
1,2-dicarba-nido-undecaborate group derived from it. In the formulas, "Me" 
designates a methyl, and "Bz" a benzyl group.

EXAMPLE 1 
The Introduction of the Benzylthio Protecting Group 
(a) 2-Benzylthio-5-bromo-4-chloropyrimidine (I) 
A suspension of 72 g of 0.242 moles (2-benzylthio)-5-bromouracil (Barrett, 
Goodman and Dittmer, J. Amer. Chem. Soc., 70, 1753, 1948) is refluxed for 
6 hours in 250 ml freshly distilled phosphorus oxychloride. The excess 
POCl.sub.3 is removed on the rotary evaporator, 250 ml ice water are added 
to the residue, and extracted with diethyl ether. The ether layer is 
washed with a saturated solution of sodium bicarbonate, dried over 
magnesium sulfate, and the ether removed on the rotary evaporator. The 
residue is distilled (170.degree. C., 0.03 mm). Yield 40 g=59%, white 
crystals, Mp 56.degree.-57.degree. C. 
(b) 2-Benzylthio-5-bromo-4-methoxypyrimidine (II) 
A solution of 17 g (0.054 moles) of I in 50 ml dry toluene are added 
dropwise to a cooled suspension of 3.25 g (0.06 moles) sodium ethoxide, 
such that the temperature does not exceed 25.degree. C. Stirring is 
continued for another 2 hours, NaCl is removed by filtering, and toluene 
is removed on the rotary evaporator. The residue is purified by 
distillation (160.degree. C., 0.03 mm). Yield 13.5 g=80%, white crystals, 
Mp 48.degree.-49.degree. C. 
(c) 2,4-Bis-(benzylthio)-5-bromopyrimidine (V) 
Benzylmercaptane (14.7 ml=0.125 moles) and 3 g (0.13 moles) sodium are 
heated to 80.degree. C. in 200 ml dry toluene and stirred vigorously for 
12-18 hours. The resulting thiolate suspension is cooled in an ice bath, 
and 14 g (0.062 moles) 5-bromo-2,4-dichloropyrimidine (Hilbert and Jansen, 
J. Amer. Chem. Soc., 56, 134, 1934) are added dropwise such that the 
temperature does not exceed 25.degree. C. The reaction mixture is stirred 
at room temperature overnight, and freed by filtration from NaCl and 
remaining thiolate. The filtrate is reduced on a rotary evaporator and 
purified by distillation (200.degree. C., 0.03 mm). Yield 16.4 g=66%, 
white crystals, Mp 66.degree.-67.degree. C. 
EXAMPLE 2 
Preparation of Boron-containing Thiourea Derivatives 
(1a) Diethanolamine derivatives of 
2-benzylthio-5-dihydroxyboryl-4-methoxypyrimidine (III) 
A 250-ml three-necked round-bottom flask, equipped with a low-temperature 
thermometer and a rubber septum, is flooded with nitrogen gas and then 
dried thoroughly with a heat gun. II (5 g-16 mmoles), dissolved in 150 ml 
dry, freshly distilled tetrahydrofurane, is injected into the RB flask 
through the septum. The solution is cooled to -100.degree. C. in a cooling 
mixture of ethanol/liquid nitrogen. n-Butyl lithium (11 ml=17.5 mmoles) of 
a 1.6 molar solution in hexane and 5 ml (18.5 mmoles) tributylborate are 
filled into syringes and cooled to -80.degree. C. in the cooling mixture. 
n-Butyl lithium is now injected into the RB flask over a period of 5 
minutes. The temperature should not rise above -85.degree. C. After 10 
additional minutes of stirring, tributyl borate is injected into the 
flask. The reaction mixture is allowed to warm to room temperature over a 
period of 1.5 hours, and evaporated on the rotary evaporator. The residue 
is dissolved in 100 ml 2 M NaOH and is extracted with 4.times.50 ml ether. 
The water layer is brought to pH=2 with concentrated HCl and again 
extracted with ether. A saturated solution of diethanolamine is added to 
the above ether extract, until no further precipitate is formed. The 
crystals are filtered and dissolved in little ethanol. Petrol ether (bp 
35.degree.-50.degree. C.) is added until a slight cloudiness develops. The 
solution is allowed to stand for 3 hours, the precipitate is filtered off 
and dried at 100.degree. C. Yield 3.5 g=63.4%, white crystals, Mp 
185.degree.-186.degree. C. 
(b) 5-Dihydroxyboryl-2-thiouracil (A) 
Five g (14.5 mmoles) of III are added slowly to a vigorously stirred 
solution of 15.5 g (58 mmoles) AlBr.sub.3 in 100 ml dry toluene. The 
reaction mixture is stirred for 5 hours at 50.degree.-60.degree. C., and 
cooled. One hundred ml ice water are added slowly. The raw product is 
filtered off, dissolved in 75 ml 1 M NaOH, and the solution extracted with 
ether. Subsequently, the water layer is acidified to pH=2 with 
concentrated HCl, the precipitate is filtered off, and washed with 
acetone. It is recrystallized from ethanol. Yield 1.2 g=48.2%, white 
crystals. Mp&gt;300.degree. C. 
______________________________________ 
Elemental analysis C.sub.4 H.sub.5 BN.sub.2 O.sub.3 S 
% 
calc. 
found 
______________________________________ 
C 27.94 28.19 
H 2.93 3.19 
N 16.28 16.21 
______________________________________ 
(2a) Diethanolamine derivative of 
2,5-bis-(benzylthio)-5-dihydroxyborylpyrimidine (VI) 
V [(6.45 g=16 mmoles, 11 ml (17.5 mmoles)] of a 1.6 molar solution of 
n-butyl lithium in n-hexane, and 5 ml (18.5 mmoles) tributylborate are 
reacted according to III. Yield 3.9 g=55.8%, white crystals, Mp 
157.degree.-158.degree. C. 
(b) 5-Dihydroxyboryl-2,4-dithiouracil (B) 
VI (6.33 g=14.5 mmoles) and 15.5 g (58 mmoles) AlBr.sub.3 are reacted as 
described for A. The product is dissolved again in 3M NaOH, extracted with 
ether, acidified to pH=2 with concentrated HCl, and filtered. Yield 1.1 
g=40.4%, yellowish crystals, Mp&gt;300.degree. C. 
______________________________________ 
Elemental analysis C.sub.4 H.sub.5 BN.sub.2 O.sub.2 S.sub.2 .times. 0.5 
H.sub.2 O 
% 
calc. 
found 
______________________________________ 
C 24.49 24.49 
H 3.08 2.79 
N 14.28 14.38 
______________________________________ 
5-Dihydroxyboryl-6-propyl-2-thiouracil and 
5-dihydroxyboryl-6-propyl-2,4-dithiouracil were prepared in an analogous 
manner. 
3. Synthesis of 4-(3-carboranylpropyl)thlyl-pyrimidine-2-thiol (E) 
4-Thiouracil (Mizumo, Ikehara and Watanabe, Chem. Pharm. Bull., 10, 647, 
1972) in dimethyl formamide is reacted with iodopropyl carborane 
(Zakharkin, Brattsev and Chapovskii, J. Gen. Chem. USSR, 35, 2149, 1965) 
to yield 4-(3-carboranylpropyl)thiyl-pyrimidine 2-ol. The chloro 
derivative is obtained with POCl.sub.3 through methods known in the 
literature. Reaction to the 2-thiol derivative with thioureau is likewise 
achieved through methods known in the literature. 
4. Synthesis of 4(5)-dihydroxyboryl-1-methylimidazolo-2-thiol (H/I) 
2-Benzylthio-4(5)-iodo-1-methylimidazole is prepared from 
2-benzylthio-4(5)-iodoimidazole (Hebner and Scholz, U.S. Pat. No. 
2,654,761, 1951) with dimethyl sulfate. This is reacted in 
tetrahydrofurane at -85.degree. C. with an equimolar amount of butyl 
lithium and then tributylborate. Further steps and removal of the benzyl 
protecting group is carried out in analogy to 
5-dihydroxyboryl-2-thiouracil. 
5. Synthesis of 4-undecahydrododecaboranylamino-pyrimidine 2-thiol (N) 
2-Benzylthio-4-chloropyrimidine is reacted in DMF with sodium 
aminoundecahydrododecaborate (Nakagawa and Aono., Chem. Pharm. Bull., 24, 
778, 1976). Removal of the benzyl group is achieved as described for 
5-dihydroxyboryl-2-thiouracil. 
In an analagous manner, 4-undecahydrododecaboranylthio pyrimidine 2-thiol 
is obtained upon reaction with disodium mercaptoundecahydrododecaborate. 
6. Synthesis of 5-nidocarboranylmethyl-1-methyl-2-thio-1,3,4-triazol (P) 
N-Methyl-thiosemicarbazide is reacted with 3-carboranyl acetyl chloride 
(Zakharkin, Chapovskii, Brattsev and Stanko, J. Gen. Chem. USSR, 36, 892, 
1966) to N-methyl-N"-B-carboranyl acetyl thiosemicarbazine. The reaction 
product is cyclized with NaOH analogous to Kroger, Sattler and Beyer (Ann. 
Chem., 643, 128, 1961). 
7. Synthesis of N,N'-thiocarbonyl-1,2-bis-(aminomethyl)nidocarborane (Q) 
1,2-Bis-(aminomethyl)-nidocarborane (Zakharkin and Grebennikov, Izv. Akad. 
Nauk SSSR, Ser. Khim. 2019, 1966) is heated slowly with carbon disulfide 
in 50% ethanol/water under nitrogen according to Chau-Der Li, Mella and 
Sartorelli (J. Med. Chem., 24, 1989, 1981). After 1 hour, an equimolar 
amount of concentrated HCl is added, and the reaction mixture is worked up 
after refluxing overnight. 
EXAMPLE 3 
Examples of Use 
1. Preparation of a Solution of 5-dihydroxyboryl-2-thiouracil for 
intraperitoneal injection 
5-Dihydroxyboryl-2-thiouracil (50.4 mg) are dissolved in 10 ml 0.12 M NaOH 
and adjusted to pH=7.8 with 0.12 m HCl. The solution is sterile filtered. 
2. Preparation of an Orally Administrable Form 
(a) Five hundred mg 5-dihydroxyboryl-2-thiouracil are dissolved in 10 ml 1 
M tris-hydroxymethylaminomethane. 
(b) Mannitol (4.5 g) and 2 g methyl hydroxyethyl cellulose are mixed for 
approximately 3 minutes, passed through a sieve of a mesh diameter of 0.8 
mm, and mixed again for 3 minutes. The obtained powder is wetted with the 
solution (a) and mixed. The humid granulate is passed through a sieve with 
a mesh diameter of 1.25 mm, dried for 2 hours at 50.degree. C. and 27 kPa 
(200 torr), passed through a sieve with a mesh diameter of 1.0 mm, and 
mixed for three minutes. The granulate obtained is mixed with 100 ml water 
p.i. and used within 5 minutes after preparation. 
3. Use of the Compound 
Balb/c mice, carrying a subcutaneously transplanted Harding-Passey melanoma 
on their hind leg, are injected intraperitoneally 5 times, with 6 hour 
intervals, with the solution prepared according to Example 3 paragraph 1 
above. The uptake in tumor is determined to 15-30 ppm with quantitative 
neutron capture radiography. The tumor is irradiated once with a neutron 
beam from a reactor, while the rest of the body is protected from thermal 
neutron by lithium fluoride embedded in epoxy resin. The growth of the 
tumor is measured daily over the course of several weeks. With a neutron 
dose of 8 MWxmin at the Medical Research Reactor at Brookhaven National 
Laboratory, it is found that the tumor does not grow in around half of the 
group treated; in the other half, tumor growth is observed only after 7 to 
10 weeks. In the absence of boron, retardation of tumor growth is found 
only in the first three weeks after irradiation.