JPS63119471A - 2-oxo-4-carboxy-pyrimidine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel compounds, processes for their preparation and compositions. In particular, the invention relates to inhibitors for the enzyme dihydroorotase and compounds for their production.

Biosynthesis of pyrimidine nucleotides is essential for the production of genetic material (DNA and RNA) in all living cells. In mammalian cells, pyrimidine nucleotides (UTP and CTP) can be synthesized from simple precursors via the de novo pathway or from truncated nucleosides (uridine and cytidine) present in the blood using the salvage pathway. Malaria parasite Plasmodium falciparum (P1asa+odium falcip)
arum) lacks a salvage pathway for the utilization of pyrimidine nucleosides and utilizes a de novo pathway to utilize UTP and c
'rp can only be synthesized.

Several useful anti-cancer drugs have been developed that inhibit specific enzymes that catalyze slow biochemical reactions in the pathways that lead to DNA synthesis. Mel-)IJ xate is a tight binding inhibitor of the enzyme dihydrofolate reductase and is used with other drugs in the treatment of childhood leukemia. 5-Fluorouracil is converted into 5-fluorodeoxy UMP within cells.
(FdUMP), deoxynucleoside monophosphate is the enzyme thymidylate synthase, so
5-Fluorouracil is used to treat certain solid tumors in humans.

Both of these drugs have selective toxicity towards cancer cells. This is because many types of cancer grow more rapidly than the body's normal cells and therefore must synthesize DNA and RNA at a faster rate. Cancer cells spend more time in stage 8 of the cell growth cycle when they are sensitive to such agents. Both methotrexate and 5-fluorouracil block DNA synthesis by directly or indirectly inhibiting thymidylate synthase. The inventors have developed a novel inhibitor of the third enzyme of the de novo pathway, dihydroorotase. Dr. Christofferson's early work produced an explanation of the catalytic mechanism of action of dihydroorotase. In a catalyzed reaction, carbamyl aspartate is converted through a transition state in which the two oxygen atoms at the 6-position of the dihydropyrimidine ring interact strongly with the zinc atom bound to the surface of the enzyme. Conceivable. After the transition state is broken and produced, dihydroorotase is given. We have synthesized sulfur and carboxy analogs of dihydroorotase that are tight binding inhibitors of dihydroorotase.

The present invention provides compounds for use as inhibitors against the enzyme dihydroorotase, which have the general formula: (1) A and B taken together =S or (
II) A is -H and B is -co- or -SR.

R1 and - may be the same or different, and -OH
; G, tri, or polypeptide group, -OR provided that R
is saturated or unsaturated C1-Il+alkyl, C1-, -
ts alkoxymethyl, or 4-alkyl-piperidinyl-alkyl i -NR-rich, provided that each R' is independently -H1 saturated or unsaturated C1~! - and R1 may be the same or different, -HS
C1-6 alkyl, hydroxyClN1m alkyl,
Hydroxy C1, @ether group, tetrahydrofuranyl,
is tetrahydropyranyl, a sugar or acetylated sugar group, hexylcarbamyl, methylglycine-N-carbonyl, or a waxy group that is hydrolyzed in vivo to 1; is -B, halo or C1-6 alkyl ;- is C
1-6 alkyl or 1-methyl-4-nitroimidazol-5-yl; and the dotted line represents a double bond which may or may not be present at the 4-5 position.

One preferred group of inhibitors believed to act by tightly binding the zinc-enzyme is formed when A and B together are -S and R□ is OR. Another preferred group believed to act by binding tightly as transition state homologues of the reaction is where A is H;
It is formed when B is -C0R1 and R1j6 and - are OH.

When R1 or R1 is alkyl, it is preferred that they are methyl. Di, tri or polypeptide groups include alanyl-glutamate, glutamyl-alanine,
and glycyl-glycine. Preferably, the -OR group is acetoxymethoxy or 4-methylpiperidinylethoxy.

When R4 or R4 is a sugar or an acetylated sugar, they can be a pentose or a hexose. A group which can be hydrolysed in vivo can be, for example, hydroxy-ethoxy-methyl. Preferably, 8 is methyl.

Particularly preferred compounds include hexahydro-2-oxo-6-thioxo-4-pyrimidine carboxylic acid (TD) 10
(referred to as HDDP); 2-oxo-1,2°3.6-tetrahydropyrimidine-4,6-dicarboxylic acid (referred to as HDDP); and (4RN.

6 R”)-2-oxo-hexahydro-4,6-pyrimidinedicarboxylic acid (referred to as HTDP).

The present invention also includes protologs, ie compounds that are converted in vivo to compounds of general formula (I), especially esters or salts. Preferred esters are nonpolar so that they are lipid soluble and can be enzymatically hydrolyzed in vivo to the free acid.

The present invention also relates to pharmaceutical compositions containing a compound of general formula (I) together with a pharmaceutically acceptable carrier. The composition can be in a form suitable for injection, oral or rectal administration or in a sustained release formulation.

The compounds are useful as anticancer and antimalarial agents.

For the treatment of cancer, it may be advantageous to co-administer an agent that also blocks the aforementioned alternative salvage pathway, preferably by including it in the same formulation as the inhibitor. Such blocking agents include dipyridamole (dip
yridamol), dilazep
) jd and nitrobenzylthioinosine.

For the treatment of malaria, it may be useful to co-administer the pyrimidine nucleotide precursor uridine or cytidine and the inhibitor, preferably in the same formulation.

Compounds of general formula (I) can exhibit keto-enol tautomerism. Therefore, the fact that a structure is designed to represent one tautomer, or the fact that a designation represents a - tautomer, should not be considered limiting. When formed in the enol form, the hydroxyl groups can be easily substituted.

The present invention also provides a method for preparing compounds of general formula C, which method comprises: (A) when A and B together are -S;
In the formula, R1, R, , R, and are as described above)
(B) When A is H and B is -CO-, the compound of formula (
(where R1 and - are as previously described) and then reducing at least one double bond.

When A is H and B is CO-, this method oxidizes the methyl group of the corresponding pyrimidine compound to a carboxyl group, preferably using the metal dissolved in a protic solvent to convert the pyrimidine ring into a dihydropyrimidine. including partial reduction to It is generally preferred to carry out the reduction with the ester rather than the acid itself.

Various methods are known for reducing ring double bonds in compounds when A and B are =S, among which are non-containing metals or metal alloys or other reducing agents and proton sources. Preferred are methods using active solvents or methods using protonated solvents. To make zinc, it is preferred to react zinc with thioorotine, an ester or other derivative of the acid or its tautomer in dry acetic acid. The free acid, ester or other derivative can then be obtained.

6-Thioorotic acid is known per se and methods for producing it have been published. However, the inventors have also invented a new synthetic method involving the chetation of derivatives of orotic acid or L-dihydrooorotic acid. Preferred reagents include phosphorus pentasulfide and 2,4-bis(4-methoxyphenyl)-1,3°2,4-dithiadiphosphohetane-
2,4-disulfide [Lawesson's reagent (Law@
sgon's+ Reagsnt)]. The chetation conditions for orotic acid itself are preferably 30 minutes to 2 hours, 60 to 100°C;
More preferably, it comprises heating at about 80° C. in ternarized phosphorus and pyridine.

Overheating and/or overreaction time may result in further reaction or decomposition.

The following examples illustrate the invention.

Example 1 (Thioorotic acid) (2-Hydroxy-6-mercabuto-4-pyrimidinecarboxylic acid or 1,2.3.6-tetrahydro-2-oxo-6-thioquitupyrimidine-4-carboxylic acid).

Orotic anhydride (2,8-dihydroxy-pyrimidine-
4-carboxylic acid) (50my) in warm pyridine (5R
1) After dissolving in the solution and adding ternarized phosphorus (215~),
The stirred mixture was heated to 90° C. for 30 minutes in an oil bath. 90
Stirring and heating at 0C was continued for an additional 30 minutes. After cooling,
Decant the pyridine solution from the undissolved residue at the bottom of the reaction vessel;
Concentrate at 40°C with a water pump vacuum bottom-tally evaporator,
The formed residue was treated with hydrochloric acid (311
The precipitated product was filtered and washed with a little water and ethanol to give an apricot-orange crystalline solid (
35■) was obtained, which consisted of a mixture of unreacted orotic acid and the desired title compound.

Example 2 (thiodihydrooorotic acid, TDE[0) (
2-oxo-6-thioxo-hexahydro-pyrimidine-4-carboxylic acid).

6-Thioorotic acid (350*) was suspended in dry redistilled glacial acetic acid (200d) with nitrogen bubbling. The mixture was stirred for 30 minutes at room temperature and stirred and heated in an oil bath at 55-60° C. for 30 minutes, by which time the orange solid had completely dissolved.

Slowly add excess zinc powder (500 #
) was added.

The sealed flask was stirred vigorously and the oil bath temperature was maintained at 60-64° C. for an additional 50 minutes, during which time the color of the solution turned dark red. The solution was then cooled to room temperature. (When it is desired to separate the TDHO as the zinc salt at this stage of the production, the solution is rapidly suction filtered through a sintered glass funnel, the filtrate is frozen in a liquid nitrogen bath and lyophilized under reduced pressure at room temperature. ).

To produce the free acid, dry and redistilled glacial acetic acid (2
Oxalic anhydride (8314) in 0d) was added to the reaction mixture. After stirring for 20 minutes at room temperature, the precipitated zinc oxalate and unreacted zinc powder were suctioned off through a sintered glass funnel, the furnace liquor was frozen in a liquid nitrogen bath, and then 0.05-0.
It was lyophilized at room temperature under a vacuum of 1 flHg. The light brown solid that finally formed was heated in an oil bath at 60° C. for 5 hours under a vacuum of 0.05 Jal IH9 to remove residual volatile organic acids. The product (3324) is approximately 65 f/TD) IO
(yield 71π) and contained about 8 dihydroorotic acid (DHO).

Example 3 Methyl L-2,6-thioxohexahydropyrimidine-4-carboxylate.

L-2,6-thioxohexahydropyrimidine-4-
Carboxylic acid (I, -dihydrooorotic acid) (1,13g
, 7.15 mmol! ') dry ethanol (loo
se). Dry hydrochloric acid gas was bubbled into the mixture for 15 minutes. The reaction mixture was heated to reflux for 1.5 hours, cooled and the solvent was removed to give a white solid. The crude product was recrystallized from acetone to give methyl L-2,6-thioxohexahydropyrimidine-4-carboxylate as colorless needles (920 to 75).

Melting point 183-185°C.

"un, m, r, (DM!30/CDC/, )
: δ10.15, brs.

NH, 7, 80, MS, NHi4.20. ddd, δ4
．． s7, 2 HE, δ4, 53.6 Hz, δ
4. Nu 3.6 R2+ 14 ; 3,73 + '
+ C0tC horse; 2.86 + dd* JS,!
18.8HztJl, 47.2Hz, R5; 2.
69 + dd, JS, s15.8Hz.

Jl, 43.6 Hz H5゜”Cn, m, r, (DMSO/CDCl!3)
: δ169.4, 166.9°151.7.51.
1. 47.8.31.4゜νwax(KBr)325
6.3092.1734゜1700.1474,143
7.1375°1345.1291.1240.121
6°1194.1028.850ffl-1 Example 4 Methyl L-2-oxo-6-thioxo-hexahydropyrimidine-4-carboxylate.

Methyl L-2,6-thioxohexahydropyrimidine-4-carboxylate (50q, 0,29 mmoz
) was added to anhydrous tetrahydrofuran (5 ml) with stirring at room temperature.
It dissolved during the process. Lauesson's reagent (71~, 0.17
11 Iffio I was added and the reaction mixture was stirred for 24 hours. The solvent was removed and the yellow crystalline residue was chromatographed on flash silica (light oil then ether),
Obtained pure thione. Recrystallization from chloroform gave methyl L-2-oxo-6-thioxo-hexahydropyrimidine-4-carboxylate as pale yellow needles (46-84%). Melting point 157-159°C.

”Hn, m, r, (CDC/3) i 1 0.4
0, brl, NH; 8, QQ, brs
, NH; 4.25. m, H4,3,74゜” CO
z”s; 3.27 and 3.13, m, 2
XH5゜(acetone): δ4.42, ffi,
H4; 3.75°a, Co, C horse; 3.33.
m, 2XH5° Example 5 2-oxo-hexahydropyrimidine-4°6-dicarboxylic acid (H'l'DP).

2-hydroxypyrimidine-4,6-dicarboxylic acid,
Hanze's Method (Journal of the American
Chemical Society Volume 89, 1967, No. 672
0-6725) to HTDP by direct hydrogenation in the presence of a metal catalyst such as rhodium.

Example 6 2-hydroxypyrimidine-4,6-dicarboxylic acid (H
DP).

4.6-dimethyl-2-hydroxypyrimidine (9,9
79,0,08 mol) of sodium hydroxide (2,5 mol)
M, 10 (1 m/) and heated to 70°C. Potassium permanganate (54,0 rd') in water (360rd')
9,0,34+! 10/ ) solution was heated to 70°C,
The above solution was allowed to soak for 1.5 to 2 hours. The reaction mixture was stirred at 70° C. for 2 hours, cooled, and filtered.

The purple color in the Oki liquid was removed with sodium metabisulfite. The furnace liquor was concentrated under reduced pressure and a cold solution of concentrated hydrochloric acid (10M, 35g) was added at 5°C to a pH of 2-3. The reaction mixture was filtered and recrystallized from water to give 2-hydroxypyrimidine-4,6-dicarboxylic acid (6,589,44
) was obtained. Melting point>250°C (decomposed).

”Hn, m, r, spectrum (D20): δ
6.31. Day.

H5゜mass spectrum ntm/z: 96 (M”-2XCO
2°8), 68 (5), 44 (100).

Example 7 Dimethyl 2-hydroxypyrimidine-4,6-dicarboxylate.

2-hydroxypyrimidine-4,6-dicarboxylic acid (6
,869,36,2 mmo/ ) in anhydrous methanol (3
acetyl chloride (5.68 g, 72
．． 4 mmo/ ) for 2 hours under reflux.

The reaction mixture was cooled and the solvent was removed to give a pale yellow solid. The crude product was recrystallized from methanol to obtain colorless needle-like crystals of dimethyl 2-hydroxypyrimidine-4,6-dicarboxylate (3,759,62%). Melting point 18
6-188℃.

1Hn, rn, r, spectrum (DMSO/CDC
J3): δ7゜84゜s, H5; 3.99,
a, 2xCO2C%.

νcoax (K111r) 3460, 3433.332
9°1747.1672.1853,1611.

1457.1442.1267.1234°1159.
1103.1044.885.783. :? ff-10
Mass spectrum/I/Ill/Z: 212 (M”
, 8%).

182 (24), 154 (100), 139 (11),
121 (25), 91 (31), 81 (14), 66 (
20).

Example 8 Dimethyl 2-oxo-1,2,3,8-tetrahydropyrimidine-4,6-dicarboxylate.

Dimethyl 2-hydroxypyrimidine-4,6-dicarboxylate (1,09,4,7 mmoj') was dissolved in warm acetic acid (17M, 70 tttl) and heated to 70°C. Zinc powder (1,59,22,9m
1lloJ) was added little by little over 1 hour. Each addition of zinc was accompanied by a purple color change in the reaction mixture.

The color gradually disappeared and then subsequent zinc was added in the same manner until completion. The formed mixture was stirred at 70°C for 30 minutes. The reaction mixture was filtered and diluted with acetic acid (17M, 2X
I Om). The liquid was evaporated to dryness under reduced pressure to give a colorless oil.

The remaining oil was dissolved in chloroform (10011Lt), filtered and the solvent was removed to give colorless crystals. The crude product was recrystallized from methanol to give dimethyl 2-oxo-1,2°3.6-tetrahydropyrimidine-4,6-dicarpoxylate (283-28) as colorless prisms.

Melting point: 177-179°C.

”Fin, m, r, suhek) /L/ (DMSO/C
DCA', ): a 8.13゜brg, NH; 7,
17brs, NH; 5.79. ddd.

'**8 5.3 Hz + Jl, Ml! 1
．． 7 H2, Js, m 1.7 Hz, 5H; 4
．． 74, dd + 'Is, s 5.3Hz, J
g, xu2.4Hz, H6; 3.73, a
, CotC bird.

νwax (KBr) 3450, 1757. 17
29°1885.1472.1457,1349°12
89.1223, 1176.1111゜1044.10
08,984,845,741crn-'.

Mass spectrum rrr/Z: 214 (M”,
596').

213(5), 155(100), 123(25).

95(71), 88(13).

Example 9 (4R only 6HM)-dimethyl 2-oxo-hexahydropyrimidine-4,6-dicarboxylate.

Dimethyl 2-hydroxypyrimidine-4,6-dicarboxylate (1,429,5,7 mmo/ ) to y'
9 (Pd/
Hydrogenated over C catalyst. The reaction mixture was filtered and methanol was removed under reduced pressure. The crude product was recrystallized from methanol/ether as colorless needles (4R', 5R'
e) -Dimethyl 2-oxo-hexahydropyrimidine-
4,6-dicarboxylate (1,27 g, 88 g) was obtained. Melting point: 178-179°C.

'Hn, ya, r, Spect/L, (DMSO/D,
O): δ2.28゜ddd+ Jfiaz, 1 to 1
4 ” l'S&!, tax(saw) 8 ”
+ 5 ”; 2, 36, ddd, Jseq,
sax 14 HE, Js8q, 4ax(saz)
4 Hz, 5'Iq;
4. l 2 * ddm J4
ax (6ax), saw 6 Hz*J4az (s
az), ssq 4 Hl, 4 a3e and 5a
x013Cn, ff1. r, Spect/L, (DMS
O/D, O): δ25.1°C5; 50.3 and 51.8, 2xCHS, C4 and C6; 151.
5. C2; 171.5.2xCOtCHs.

νmax (KBr) 3249.3099, 2963°1
751.1895, 1533, 1450°! 253.
1202.1042.817,775cr+g-'.

Quality f/lx vector I! +/'; 216 (" +
109f; ) +157 (Zoo), 114 (73
), 97(34).

82(23).

Example 10 General hydrolysis ester (Q, 5 mmoJ) was heated to reflux in a solution of sodium hydroxide (IM, 2.5 m/) for 30 minutes,
Cooled. The reaction mixture was filtered and purified with salt [(10M) p
Acidified to H3-4 and lyophilized the solvent. The mixture was recrystallized from water.

(A) (4R Homare, 6R') -2-oxo-hexahydropyrimidine-4,6-dicarboxylic acid (HTD
P).

Dimethyl 2-oxo-hexahydropyrimidine-4,6
- The dicarboxylate is hydrolyzed by the above-mentioned membranous properties as white crystals (4R%).

6R')-2-oxo-hexahydropyrimidine-4,
6-dicarboxylic acid (36) was obtained.

1Hn, rn, r, spectrum (D20):
δ2.10, ddd.

JIJLX, $11 (l l 3.5 H
z, JSaz, 4az (gaz) 5
．． OH2, 5eq'.

4, 1 3 * dd + J4az (Iax
), Iax s, OHz, J4az (aax),
5eq5, QHz, 4ax and f3ax0 mass spectrum m/z: l44 (M-co2.
IK).

100 (6), 71 (4), 58 (
5), 44 (lOO).

(B) 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid (HDDP).

Dimethyl 2-oxo-1°2.3.
Hydrolysis of 6-tetrahydropyrimidine-4,6-dicarpoxylate produces 2-oxo-1 as colorless needle-like crystals.
, 2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid (50π) was obtained.

'Hn, lII, r, spectrum (D20):
δ6.08. d.

's, a 5. OH” +H5i4.9 0 +
d+ J@, s 5.0”+H6° mass spectrum m/z: 158 (M-co, 0.4π),
142(0,1), 126(0,1), 100(0,4
), 90(5), 56(15), 48(80).

45 (100), 44 (89).

(C) 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid (IIIDDP).

2-hydroxypyrimidine-4,6-dicarboxylic acid (2
a 91nf, 1.30 armol was suspended in acetic acid (17M, 60m) at 70°C with stirring.

Zinc powder (6004,9,17 mmoJ) was added portionwise over 30 minutes and the mixture was stirred at 70° C. for 1 hour and then cooled to room temperature. Oxalic acid (200ml) in acetic acid (10ml)
q, 2.22 a+moj') was added and the reaction mixture was left for 5 hours. The mixture was filtered and the filtrate was evaporated to dryness. The residue was dissolved in hot water 5-, passed through,
Cooled to 4°C for 4 days.

2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid (58 rng
, 24%).

Example 11 General method for producing amides from esters.

The ester (l armol) was dissolved in 20 g of methanol and cooled to 0-5<0>C. 40 minutes while stirring the mixture.
Ammour was bubbled into the solution for a minute.

The reaction vessel was stoppered and the mixture was allowed to warm to room temperature and left for 16 hours. The reaction mixture was filtered and washed with ethanol to obtain a white solid.

(A) 2-hydroxypyrimidine-4,6-dicarboxamide.

2-hydroxypyrimidine-4,6-dicarboxamide was obtained by ammonolysis of dimethyl 2-hydroxypyrimidine-4,6-dicarboxylate according to the general method described above.

1Hn, m, r, spectrum/l/(D, O): δ7
．． 51,8°H5°'Cn, m, r, spectrum (D!0): δ171
．． 5°171.4, 163.6. C2, C3 and C5
゜2xCONH,; lO4,9,C5゜Mass spectrum m/z: 182 (M", 359 ().

139 (100), 93 (20), 67 (40).

44 (62).

(B) (Both 4R and 5R)-2-oxo-hexahydropyrimidine-4,6-dicarboxamide.

Dimethyl 2-oxo-hexahydropyrimidine-4,6-dicarboxylate was ammonolyzed by the general method described above to give (4R', 6R'')-2-oxo-hexahydropyrimidine-4,6-dicarboxylate as a white solid. The amide (72) was obtained.

Example 12 [(1,0mmo7F), bromoethyl acetate (1
, 1armol') and anhydrous triethylamine (1
, 2 mmal) of anhydrous acetonitrile (10x/)i
3 and anhydrous acetone (10x/) at room temperature.
Stirred for 8 hours. The reaction mixture was filtered and the solvent removed. The residue was recrystallized from acetone to obtain colorless crystals of triethylamine hydrobromide.

Example 13 Production of glycylglycine ethyl ester.

Pyrimidinecarboxylic acid (3,00 mmol) in anhydrous dimethylformamide (25 tug), IIDQ (
2-Imbutoxy-1-isobutoxycarbonyl-1,
2-dihydroquinoline) (3,75 mmol) Glycylglycine ethyl ester hydrochloric acid [(3,75 mmo/
) g and anhydrous triethylamine (3,75++mol
The solution was stirred at 50-55°C for 36 hours. The solvent was removed under reduced pressure and the residue was dissolved in ethanol (25x/) and poured into hydrochloric acid (IM, 200m/). The precipitate was filtered, dried and purified by chromatography (flash silica).

This manufacturing method was commonly used for the synthesis of heptides, dipeptides and polypeptides.

Example 14 Production of pyrimidine dipeptide acid.

Pyrimidine glycylglycine ethyl ester (1, Om
mo/ ) was dissolved in a solution of hydrochloric acid in glacial acetic acid (LM, 6 ml). The solution was stirred at room temperature for 2 hours and then evaporated with HC under reduced pressure.
I! was removed. Acetic acid was removed by lyophilization and the residue was washed with anhydrous ether to obtain white crystals. Update this! '
Purified by 1PLC.

Example 15 Production of N-hexylcarbamoylpyrimidine.

Pyrimidine (1,0 mmo/') and hexyl isocyanate (1,5 mmo/') were heated in pyridine (4 rxl) at 90 °C for 1 h, cooled to room temperature, and the solvent was removed under reduced pressure at 50 °C. Heat the residue with ethanol (5 grams)
The solution was dissolved in water, filtered, and cooled to 0-4°C for 24 hours to obtain a crystalline product.

Example 16 Preparation of N-methoxycarbonylmethylcarbamoylprimidine.

These compounds were made by the general method described above using 1-methoxycarbonylmethyl isocyanate.

Test 1 (Purification of dihydroorotase) Dihydroorotase was purified by established methods from a mutant hamster cell line that overproduces the target enzyme by more than 100 times [New York, Academic Press, 1978, Methods In.・P. in Enzymology (edited by P. Ni. Holafy and M.E. Johns)
F. Coleman, D. P. Saratre and G.
Earl Stark's paper; Journal of Biological Chemistry 1977, Volume 252, No. 63
(See the article by P.F. Coleman, D.P. Saratre, and G.R. Stark, pp. 79-8385). The purified dihydroorotase was diluted to 30 (V/
m) Dimethyl sulfoxide, 5% (v/v) glycerol, 50+aMKC/14mML +, glutamine,
4mM L-aspartate, Q, lmM EDT
It was stored at -70°C in a solution containing A and lmM dithiothreitol.

Test 2 (Inhibition generating ability of TDHO) Dihydroorotase catalyzed the conversion of N-carbamyl-L-aspartate to L-dihydroorotate. In order to evaluate the effect of this potential inhibitor of 9), the rate of conversion of L-dihydroorotate (7) to N-carbamyl-L-aspartate catalyzed by dihydroorotase was evaluated. Measured in the presence of a range of concentrations. 'I'DF! For tight binding inhibitors such as O, extremely low concentrations were sufficient to terminate all dihydroorotase activity. To demonstrate the ability of TDHO to cause inhibition, the following experimental results are presented. The assay mixture for dihydroorotase activity was 25 μl of 5 mM K. Hepesba Sofer PF! 7.4, 59g (v
/v) 'f'J cea -/L/, 12.5 μM L-(14C] dihydroorotate (specific activity 47.1
01/mo/ ), and TDHO concentrations ranging from 0 to 1,5 μl containing a 30-fold molar excess of ZnClt. Catalysis was initiated by the addition of dihydroorotase 39 n9. Established Methods (Analytical Biochemistry - 1978, Vol. 100, No. 18)
rl, rl, in the presence of various concentrations of TDHO using
The rate of conversion of -C14C]dihydroorotate to N-[14C]carbamyl-L-aspartate was determined.

The assay mixture lacking TDHO had the fastest reaction rate, whereas '! In the presence of the highest concentration of HO
This was the lowest speed measured. TDH the obtained data
Plotted as the reciprocal of the reaction rate (1/V') against the concentration of O, then the TD from the enzyme-TDHO complex
A value for the dissociation constant (Ki value) for HO can be obtained.

From the data, a Ki value of 53 nM was calculated, which indicates that the target enzymes dihydroorotase 3 and T
This shows a very strong interaction between DHO. The results also show that ZnC1 alone (at the same concentration) has only a moderate inhibitory effect, while the free acid of TDHO (absence of Zn''+) has a 4.5
It was also shown to be a less effective inhibitor with a Ki value of μM.

Test 4 (Inhibition generation ability of HDDP) The assay mixture for dihydroorotase was 25μm,
5% in 5mM K. hepes buffer pH 7.4
(v/v) glycerol, 12 μM I, -C14C)
Dihydroorotate (specific radioactivity 47.IC1/mo/
), and HDDP concentrations ranging from 0 to 0.5 μM. Contacting was initiated by adding 39n9 dihydroorotase. Measurements were made in the same manner as in Test 3 in the presence of different concentrations of HDDP.

From the data, we calculated an xi value of 0.48μ, which corresponds to the target enzyme dihydroorotase and H
showed a strong interaction between DDP.

Claims (1)

[Claims] 1. General formula (I) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ [In the formula, (i) A and B together are =S, or (ii) A is -H and B is −COR_2 or −
SR_3; R_1 and R_2 may be the same or different.
OH, di-, tri- or polypeptide group, -OR where R is saturated or unsaturated C_1_ to_1_8 alkyl, C
_1_ to _1_6 alkoxymethyl, or 4-alkyl-piveridinyl-alkyl, -NR'R', but each R'
each independently -H, saturated or unsaturated C_1 to C_
1_6 alkyl, or a group that can be hydrolyzed in vivo to hydroxy; R_3 and R_4 may be the same or different;
-H, C_1_~_6 alkyl, hydroxyC_1~C
R_5 is a _1_6 alkyl, hydroxyC_1_-_6 ether group, tetrahydrofuranyl, tetrahydropyranyl, a sugar or acetylated sugar group, hexylcarbamyl, methylglycine-N-carbonyl, or a group that can be hydrolyzed in vivo to -H is -H, halo or C_1_-_6 alkyl, R_6 is C_1_-_6 alkyl or 1-methyl-4-nitroimidazol-5-yl, and the dotted line is present or absent in the 4-5 position. a double bond which may be present] for use as an inhibitor against the enzyme dihydroorotase. 2. Claim 1 in which A and B together =S
Compounds described in Section. 3. The compound according to claim 1 or 2, wherein A is -H and B is -COR_2. 4. The compound according to claim 3, wherein R_1 and R_2 are each methyl. 5. The compound according to any one of claims 1 to 4, wherein R_3 is -H and R_4 is ribosyl. The compound according to claim 1, which is 6,2-oxo-6-thioxo-hexahydropyrimidine-4-carboxylic acid. Claim 1 which is 7,2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
Compounds described in Section. The compound according to claim 1, which is 8,2-oxo-hexahydropyrimidine-4,6-dicarboxylic acid. 9. A compound according to claim 1 which is convertible in vivo into a compound according to claim 6, 7 or 8. 10. A composition containing a compound according to any one of claims 1 to 9 together with a pharmaceutically acceptable carrier. 11. Use of a compound according to any one of claims 1 to 9 for the treatment of cancer. 12. Use of a compound according to any one of claims 1 to 9 for the treatment of malaria. 13, (A) When A and B together =S, the formula (
II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (wherein R_1, R_3, R_4 and R_5 are the same as in claim 1) is thiated, and then at least one double bond is removed. (B) When A and B together =S, formula (IV)▲
There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1, R_3, R_4 and R_5 are the same as in claim 1) The compound of (C) A is H and B is -COR_2, the compound of formula (III) ▲ has a mathematical formula, chemical formula, table, etc. ▼ (wherein R_3, R_4 and R_5 are the same as in claim 1) is thiated;
Then, at least one double bond is reduced.
I) Method for producing the compound. 14. The thiating agent in (A) is phosphorus pentasulfide or 2,4-bis(4-methoxyphenyl)-1,3,2,
14. The method according to claim 13, which is 4-dithiadiphosphethane-2,4-disulfide.