Synthesis of some non-antimonial compounds bioisosteric to praziquantel

525

Eur J Med Chetn (1995) 30,525-529

0 Elsevier,Paris

New products

Synthesis of some non-antimonial

compounds bioisosteric to praziquantel

HH Hassaneinl, HM El Nahalz, FR Gerges2 IDepartment

of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini 2Department of Parasitology, Theodor Bilharz Research Institute, Giza, Egypt

Street, Cairo;

(Received5 July 1994;accepted27 February 1995)

praziquantel

bioisostere

/ scbistosomicide

/ pyrido[l,2-c]pyrimido[5,4-elpyrimidine

/ Sch&osonuzmunsoni

Introduction Schistosomiasis is a major global health problem and yearly there are hundreds of millions of people exposed to the disease [l]. In Egypt infection with schistosomiasis is a common national health problem. The traditional established schistosomicides are effective but all share the disadvantages of lengthy treatment, parenteral administration, high toxicity and narrow spectrum of activity. There is a need for safe, orally active and broad spectrum antibilharzial agent. Praziquantel, 2-cyclohexylcarbonyl-1,2,3,6,7,1lbhexahydro-4H-pyrazino[2,1-alisoquinolin-4-one I [2, 31 is a broad spectrum orally active schistosomicide derived from a novel nitrogen bridgehead heterocyclic system [4-71. Extensive pharmacological investigations have revealed that, in contrast to antimonial and organic phosphorus antibilharzials, praziquantel has potent but non-significant side effects at therapeutic doses [8, 91. This finding opened the way to the chemistry of bridgehead compounds as antibilharzial drugs. In this field the ideal criteria of praziquantel prompted us to synthesize some nitrogen bridgehead compounds 6 (a R = C,H,, b R = (;H,, c R = CH,) structurally related to praziquantel. In the design of our new condensed system we are interested in certain features, in particular that the isoquinoline ring is substituted by the bridgehead pyrido[l,2-clpyrimidine and that the pyrazine ring is replaced by a ring equivalent to the hydropyrimidine ring system with an inverted carboxamide function [lo].

I

6

Fig 1. Structures of praziquantel I and compound 6 (a R = C,H,, b R = C,H,, c R = CH3).

Chemistry The methods used to prepare compounds 6a,b,c are shown in scheme 1. The gem-dinitrile 1 [l l] reacted with isocyanates in an inert solvent to afford the nitrogen bridgehead iminonitrile pyrido[1,2-clpyrimidine 2. Metal hydride reduction of 2 gave rise to the corresponding enamine 3 [12]. Treatment of 3 with an acid chloride (COCI),, afforded the tricyclic 3-chlorocarbonyl-5-alkyl-9,10,11,1 la-tetrahydro-8H-pyrido[1,2-c]pyrimido[5,4-elpyrimidine-1,6-(W,-dione 4 [13]. The intermediate 4 was refluxed in absolute ethanol to give the ethyl carboxylate, ethyl-5-alkyl9,10,11,11a-tetrahydro-8If-pyrido[1,2-c]pyrimido[5,4-elpyrimidine-1,6-(W,SH)-dione 5. The designed compounds 6 were prepared either from the acid chloride (Route a) or from the ethyl ester (Route b) by refluxing with the appropriate amine. In the above

526 TabIe

I. Physical

data for compounds

Compound

5

MP W)

6a,l

-NH2

248-249

6a,2

-NHNH2

232-233

6aJ

-NHCH,

228-229

6a,4

-NW,),

187-188

f&5

-NH&H,

162-163

6a,6

-N

Molecular formula

3

‘;,.b.C-

6a,7

-N

3

Scheme 1. R = CH,, GH, or C,H,.

reaction it was found that Route b gives a better yield than Route Q. The structures of the present compounds were established by elemental and spectral data (IR, IH-Nh4Ft and mass spectrometry; tables I, II and III). study

Four selected compounds of the pyrido[l,2-clpyrimido[5,4-elpyrimidi nucleus 6a,7, C&,8, 6b,7 and 6c,7 were screened for antibilharziaI activity in mice pre-infected with Schistosoma mansoni. The measure of evaluation are worm load, oogram and ova count. The results are listed in table IV and figures 24. A study of the acute toxicity of the derivatives was also made [12]. Results and discussion From the data recorded in table IV, it can be observed that the tested compounds are significantly active as

-N

L-lo MO-161

w9

6a,lO

6a,ll 6a,12

160-161

/

6a,8

Phannacekqgical

6a.

Ii -N-

O

215-216

-NH-CH2

197-198

-NH

175-176

antibilharzial agents. The responses are varying in degree of potency and stage of activity. Compound 6c,7 was found to be the most potent in three criteria of evaluation as compared with the control and reference antibiiharzial drug.

527 Table

II. Physical data for compounds6b.

Table

III. Physical data for compounds6c.

0

Compound

MP W)

6b,l

-NH,

6b,2

-NHNH,

Molecular formula

312-313

Compound

MP (“C)

6c,l

-NH,

291-292

6c,2

-NHNH,

230-231

-NHCH3

240-24 1

a,4

-N(CW

217-218

6c,4

-N(W),

6b,5

-N

24&241

6c,5

-N

3 n

6b,6

-N

6b,7

-N

-NHCH,

-N

6c,7 3 H

6b,8

-N-

O

i-Jo

H N

205-206 215

Q

212-213 6c,9

.n

230-231

180-181

a,9

6c,lO

It is also worth noting that the new compounds are non-toxic and the toxicity decreases with relatively lengthy alkyl at position 5 (R = C,H,). The LD,, values are in the range 2150-3100 mgkg body weight orally in mice. Experimental

3

N 3

172-173 6c,8

195-196

n

6c,6

LIP

Molecular formula

protocols

Chemistry All melting points are uncorrectedandwere determinedby the open capillary method using Griffin melting point apparatus. Microanalysis was carried out at the Microanalytical unit, Faculty of Science, Cairo University. Infrared spectra (KBr

191-192

pellets)were recordedon Shimadzuinfrared spectrophotometer IR 435. iH-NMR spectrawere recordedon T90 MHz NMR usingCDCI, asa solvent andTh4S asa standard. 2-Alkyl-4-cyano-3-imino-.5,4,7,8-tetrahydro-3H-pyrido[l,2-c]pyrimidin-1-(2H)-ones2. 2-Dicyanomethylidene-2,3,4,5-tetrahydro-~,6H-pyridine1 was preparedfrom the correspondinglactim ether. Compound 1 addedto isocyanatesin equimolarratio to give 2. 2-Alkyl-3-amino-4-cyano-2,4a,5,6,7,8-hexahydro-lH-pyrido[1,2-c]pyrimidin-I-one 3 lminonitrile 2 (0.01 mol) was suspendedin ethanol (20 ml). Sodium borohydride (0.006 mol) was addedand the reaction mixture wasleft overnight. The mixture was treatedwith drops of water, and the separatedcrystals washedwith water and crystallisedfrom ethanol.

528 Table IV. Antibilharzial Group

activity. __.-_

Mean number

Oogram (stage)

Ova count/gram

tissue

0fworm.s .___-

(n = 10) 1st 1. control 2. Treated PZ

10.40 + 1.19 0

3rd

4th

28.55 2 0.98

2.67 + 0.50

2nd

5.08 + 0.65 12.04 f 1.29

Mature 36.60

2 1.96

0

0

0

0

10.72 2 10.26

0

0.14 2 0.1

3.47 + 2.48

3. Treated 6a,7

3.50 2 1.19

0

0

4. Treated 6a,8

7.40 + 1.4

0

8.60 t 3.7

21.40

2 6.53

5. Treated fib,7

4.20 t 1.24

0

2.80 t 1.87

23.62

+ 6.46

6. Treated 6c.7

0.20 2 0.13

0

0

5-Alkyl-3-chlorocarbonyl-9,I0,ll,lla-tetrahydro-8H-pyrido[1,2-c]pyrimido[S,4-e]pyrimidine-l,6-(2H,5H)-dione

0 8.80 + 4.77

0

4.

To a mixture of 3 (0.05 mol) and dry benzene(10 ml), oxalyl chloride (0.05 mol) was added and the mixture was kept at ambient temperaturefor 3h. The solvent was then distilled under vacuum and crystalline product was separated;yield 96.5%. Ethyl-5-al~l-9,10,Il,lla-tetrahydro-8H-pyrido[l,2-cJpyrimido[5,4-e}pyrimidine-1,6-(2H,5H)-dione 5

Acid chloride 4 was treated with ethanol (30 ml) and refluxed for 2 h. The mixture was then allowed to standfor 24 h and excesssolvent wasremoved in vacua. Crystalsseparatedfrom ethanolwith nearly quantitative yield.

0

Dead 13.06 2 1.43

89.09 ?r 3.03

23 354.80

?r 1.85

13 471.302

+ 8.43

16 384.17

+ 5.07

26.28

22.38

+ 5.70

42.40~

100

11.60

Intestine

f 4733.78

5288.40 2 1014.71

96.39

23.72

0

Liver

1565.79 f. 4283.49

36 993.30

+ 11457.70

2996.2Oi789.10 9388.4021999.67 5061.00~

1316.03

7391.41 + 1875.55

5432.052

1507.74

6613.31 + 2361.49

3358.96 f 1136.19

General methods for preparation of N-alkyl-5-a&l-9,1O,ll,llatetrahydro-BH-pyrido[1,2-c]pyrimido[5,4-e]pyrimidine-l,4(2H,SH)-diones 3-carboxamides ba, b and c

To a mixture of 5 (0.001 mol) and ethanol(15 ml) two equivalents of the appropriate amine were added. The mixture was stirred until complete dissolution.The solution was then refluxed for 4 h, the solvent was removed under reducedpressure,and the separatedcrystals were filtered and crystallisedfrom ethanol.

Route A.

Route B.

If no crystalline product obtainedfrom Route a, the oily masswas triturated with water, extracted with CHCl,, and the chloroformic extract washed with dilute H$O,. The chloroformic extract wasdistilled and the residuewastriturated with ether. data. The infrared spectrumof 6a, 9 revealed the following bands:3400-3200(NH CO), 2950 (CH aliph), 1655 Spectral

Fig 2. Antibilharzial activity (worm load) of the tested compounds.0 mean; n standarderror.

Fig 3. Antibilharzial activity (oogram) of the tested compounds. + control, t PZ, x 6a,7 + 6a,8 w 6b,7 0 6c,7.

529

1

Ova countlgram of tissue A piece of liver or intestine was digested in 5% KOH, after being weighed and incubated at 37°C for 24 h. The digest was well shaken and 3 samples (100 pm each) of the digest were examined under the low power of the microscope to count the ova in each aliquot. The number of ova in each group was counted to obtain the mean number of ova/gram tissue [14,15]. The more potent the drug, the lower the number of ova per gram of tissue. Oogram The methods of Pelligrino et al [16] were used. Three, 1 cm long pieces of the intestine were cut off, opened and cleaned. They were then pressed between a slide and a cover slip and examined under the low power of microscope to determine the stages of development of the eggs. According to the size of the embryo inside the egg shell, it was classified into the following stages: 1, 2, 3, 4 mature and dead eggs stages. All of these stages were counted separately to get the percentage of each stage. The more efficient the drug the more the disappearance of the first four stages and the more the presence of mature and dead eggs.

Fig 4. Antibilharzial activity (ova compounds. 0 liver; I intestine.

count)

of the tested

(CO NH) cm-t. The mass spectrum of 6a,9 had an ion peak at m/e 402, which is in accord with the molecular weight. The spectrum revealed the following fragments: 402 (M+, 1.78), 389 (4.72) 348 (27.9) 347 (57.4), 319 (17.7), 275 (11.44) 263 (21.2), 225 (67.89), 141 (41), 77 (100). The tH-NMR spectrum (CDCl,, 6 ppm) of 6c,6 had the following signals 6 (ppm) 1.6-2.2 (m, 6H, Cg, C,,, C,,), 2.8 (m, 2 x 2H, CH2NCHZ), 3.35 (s, 3H, NCH3), 3.7 (m, 2 x 2H -CH,OCH,), 4.3 (t, 2H, C,). Antibilharzial

Acknowledgment We are grateful to F Yousif, Director of schistosom biological supply program, Theodor Bilharz institute, for help and cooperation.

References 1

activity

Experimental animals and method Clean laboratory bread Albino mice, CD, strain of both sexes were infected with 50 S mansoni cercariae each. Eight weeks post infection, the animals were divided into six groups (10 mice each). Group 1 were left without treatmem as a control. Group 2 were treated with praziquantel at a dose of 500 mg/kg body weight and repeated the next day. The reference drug was suspended in Cremophore El and given orally. This regimen was used for all tested compounds. Groups 3, 4, 5 and 6 were treated with drugs 6a-7,6a-8,6b-7 and 6c-7, respectively at a doses of 500 mg/kg body weight for two consecutive days. All the animals were sacrificed 10 weeks post infection (two weeks post treatment) and the criteria, worm load, oogram and ova count, were investigated in each group. The recorded data were compared with the control and reference groups 1 and 2. Worm load The worms obtained from each animal by perfusion two weeks after treatment were counted; the mean represents the worm load. The smaller the worm load the more potent the drug.

6 7 8

9 10 11 12 13 14

15 16

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