Eur J Med Chem (1992) 27,851-855 0 Elsevier,
Chemotherapeutic agents XXV: synthesis and leishmanicidal activity of carbazolylpyrimidines* VJ Raml, N Haquel, PY Guru* ‘Medicinal Chemistry Division, Lucknow; 2Parasitology Division, Central Drug Research Institute, Lucknow 226001, India (Received
1991; accepted 13 May 1992)
carbazole / pyrimidine / pattern recognition / leishmanicidal / macrophage
Introduction Being an integral part of the skeleton of DNA and RNA bases and of many bioactive substances, pyrimidines are diversely involved in living organisms and many aspects of chemotherapeutics. This prompted us to design pyrimidine molecules with a desired functionality at a specific position for molecular recognition. The importance of pyrimidines as leishmatricides [l-5] aroused considerable interest in developing compounds which could not only show antileishmanial activity but also effectively modulate the natural defense of the host and restore the impaired immune functions. Based on a pattern of recognition approach, it was considered worthwhile to synthesize compounds with a molecular makeup in which pyrimidine is linked with carbazole through a C-C bond in anticipation of their having an improved leishmanicidal activity. N-Ethylcarbazole-5-carboxaldehyde was considered as an appropriate precursor for the synthesis  of 5-cyano-6-(N-ethyl-carbazol-3-yl)-2-thiouracil 1. Alkylation of 1 with an equimolar amount of an alkyl halide at 0-5°C in DMF, using potassium carbonate as a base yielded 2-alkylthio-3,4-dihydro-6-(N-ethylcarbazol-3-yl)-4-oxopyrimidine-5-carbonitriles (2a-j) as a major product, but reaction with two equivalents of alkyl halide at ambient temperature exclusively gave 2-alkylthio-3-alkyl-3,4-dihydro-6-(N-ethylcarbazol-3-yl)-4-oxopyrimidine-Scarbonitriles (3a-c). Interaction of 1 with 1,2-dibromoethane and 1,3dibromopropane separately in DMF in the presence of potassium carbonate provided bicyclic products 6a, b. *CDRI Communication
Under similar reaction conditions reaction of 1 with 2,4-dinitrochlorobenzene or picryl chloride yielded 3cyano-4-(N-ethylcarbazol-3-yl)-7-nitro/7,9-dinitropyrimido[2,1-blbenzothiazol-2-ones 7a, b. The possibility that a linearly cyclized product such as 3-cyano2-(N-ethylcarbazol-3-yl)-7-nitropyrimido[2,1-b]benzothiazol-4-one (7a) was ruled out, as the H-6 proton in NMR resonated in downfield at 6 9.18 (d, J = 3 Hz) due to the anisotropic effect of the carbazole ring and deshielding effect of the NO, group. Irradiation of this resonance frequency (6 9.18) enhanced the intensity of H-l’ proton. Chemical shifts at 6 8.54 (dd, J = 3, 8.9 Hz) and 9.01 (d, J = 9 Hz) ‘were unambiguously assigned for H-8 and H-9 protons while peaks at 6 8.97 (d, J = 3 Hz) and 8.26 (dd, J = 3, 9 Hz) ascertained for H-4’ and H-2’ protons of the carbazole ring. Signals at 6 7.71, 7.85 and 8.3 with coupling constant of 9 Hz each were assigned for H-8’, H-l’ and H-5’ protons respectively. Irradiation of the signal at 6 7.85 (H-l’) gave strong noes for H-2’, H-4’ and H-6 protons. Halogenation of 2-benzylthio-3,4-dihydro-6-(/Vethylcarbazol-3-yl)-4-oxopyrimidine-5-carbonitrile (2i) with phosphoryl chloride provided 4 which was vulnerable to a nucleophilic substitution with primary and heterocyclic secondary amines to yield 5a-d. Stirring a mixture of 3 with little excess of one equivalent of 80% hydrazine hydrate provided 6-(Nethylcarbazol-3-yl)-3,4-dihydro-2-hydrazino-3methyl-4-oxopyrimidine-5-carbonitrile 8. Biological
The test system applied to assess the anti-leishmanial activity in vitro was similar to that of Neal et al , Chang  and Bhatnagar et al . Macrophages
isolated from the peritoneal cavity of cotton rats infected with amastigotes of L donovani were used. These macrophages were suspended (106 macrophages/ml) in medium containing 10% fetal calf serum. One ml of this was then seeded in each lighton tube. The following day the medium was replaced by fresh medium containing 3 x 106 promastigotes/ml. The required amount of drug (100 pg) was dissolved and a suspension was prepared in the complete medium. Older medium was replaced the following day by the drug containing medium. After 2 days the medium was again removed and replaced with the fresh medium containing drug in the same concentration. Another 2 days after the second administration, the lighton tubes were stained with Giemsa. The average number of amastigotes per macrophage in treated as well as untreated control was calculated. For each test, 3-4 tubes were prepared and the data of these two replicates were compiled for calculating the mean percentage inhibition. The antileishmanial activity in vitro was expressed as percentage inhibition and compared with standard drug stibanate (sodium stibogluconate). The leishmanicidal activity of 12 screened compounds 1, 2d, 2d, 2e-j, 3c, 6a, b is shown in table I. All the compounds were screened at 100 pg/ml concentration. Results and Discussion Among the 12 screened compounds only 2d and 2f exhibited maximum 59% of inhibition while 2h was effective only up to 42%. Some of the compounds such as 2j, 3c and 6a demonstrated moderate activity while the other compounds were either toxic or poorly active. Table I. Antileishmanial activity of compounds of Leishmania donovani in vitro.
Activity % inhibition (at 100 /@ml dose)
Toxic 42 26
2i %i 2
It is evident from the screening data that the nature and type of substituent of position 2 plays a crucial role in leishmanicidal activity. The presence of 3 carbon unit attachment or substituent of similar length at sulphur in 1 is the minimum requirement for displaying anti-leishmanial activity. The lower activity of 2b is presumably due to the reduction in length of 3 carbon units of cyclopropylmethyl substituent. Experimental
Melting points were determined in an open capillary and are uncorrected. The comuounds were analvzed for C. H, and N in micro analytical section of CDRI. IR- spectra were obtained from a Per&r-Elmer 157 spectrometer and mass spectra were recorded on a Jeol JMS D-300 suectrometer. 1H NMR suectra were recorded on a Perkin-Eimer R-32 (90 MHz)* with tetramethyl silane as an internal standard.
5-Cyano-6-(N-ethylcarbazol-3-y1)-2-thiouracil Z This was prepared from N-ethylcarbazol-3-carboxaldehyde thiourea and ethyl cyanoacetate as described by Ram [lo], mp 265”C, yield 56%. Anal C,,H,,N,OS (C, H, N), MS: m/z = 346, tH-NMR (CDCl, + DMSO-d,): 6 (ppm) = 1.34 (t, 3H, CH,), 4.30 (q, 2H, CH,), 7.02-7.6 (m, 4H, Ar-H), 8.02 (t, 2H, Ar-H), 8.59 (d, lH, Ar-H).
3.4-Dihvdro-6-(N-ethvlcarbazol-3-vl)-2-methvlthio-4-oxopjrimidine-5-caibonitrile 2a _ A solution of 1 (1 P. 2.9 mmol) in DMF (20 ml) was stirred with potassium &b&ate (0.4 g,‘2.9 mmol jand cooled to 0°C. Methyl iodide (0.42 g, 2.96 mmol) was added dropwise to the cold solution with stirring. After complete addition, it was further stirred for 2 h at this temperature and stirring was continued for an additional 2 h at ambient temperature. The reaction mixture was diluted with water and filtered. The filtrate was neutralized with acetic acid, yellow precipitate thus obtained was filtered, washed with water and crystallized from DMF-methanol, yield 0.5 g (47%), mp > 29O’C. Anal C,,H,,N40S (C, H, N), MS: m/z = 360, IR (KBr): v (NH) = 3400 cm-t, v (C=N) = 2250 cm-i, v (C=O) = 1660 cm-l. Other compounds 2b-j were prepared similarly and are shown in table II with their relevant data.
A mixture of 1 (1 g, 2.9 mmol), methyl iodide (0.9 g, 6.3 mmol) and notassium carbonate (1 a. 7.24 mmol) in 20 ml of DMF was s&red for 3 h at room temperature. tie reaction mixture was poured into water with stirring. The white precioitate thus obtained was filtered and crvstallized from DMF. yikld 0.36 g (33%), mp 254°C. Anal C;,HrBN,OS (C, H, N); MS: m/z = 374, IR (KBr): v (C=N) = 2250 cm-l, v (C=O) = 1680 cm-l. iH NMR (CDCI,): 6 (ppm) = 1.45 (t, 3H, CH,), 2.77 (s, 3H, SCH,), 3.57 (s, 3H, NCH,), 4.38 (q, 2H, CH,), 7.23-7.58 (m, 4H, Ar-H), 8.08-8.43 (m, 2H, Ar-H), 8.94 (d, lH, Ar-H).
3-Ethvl-2-ethvlthio-6-(N-ethvlcarbazol-3-yl)-3,4-dihvdro-4_ oxopy>imidi&-S-carbonitrile jb
This was DreDared from 1 and ethvl iodide as described in the preceeding experiment in 20%* yield, mp 194°C. Anal C,,H,,N40S (C, H, N), MS: m/z = 402, IR (KBr): v (CH) =
8.53 Table II. Yields
data of 6-(IV-ethylcarbazole-3-yl)-2-thiosubstituted
Formula* (mol wt)
(C=O) = 1660 cm-l, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-l TFA 6 (ppm) = 1.23 (t, 3H, CH,), 1.3 (t, 3H, CH,), 3.24 (q. 2H, SCH,), 4.15 (q, 2H, NCH,), 7.05-7.35 (m, 4H, Ar-H), 7.68-7.85 (m, 2H, Ar-H), 8.35 (s, lH, Ar-H)
analyses (C, H, N) within
(C=O) = 1660 cm-t, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-t CDCl, 6 (ppm) = 1.44 (t, 3H, CH,), 4.00 (d, 2H, 3-CH,), 4.32 (d, lH, CH), 4.42 (q, 2H, CH,), 5.10-5.50 (m, 2H, CH,), 7.04-7.70 (m, 4H, Ar-H), 8.03-8.35 (m, 2H, Ar-H), 8.86 (d, lH, Ar-H)
(C=O) = 1660 cm-t, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-t CDCl, + DMSO-d, 6 (ppm) = 1.21 (t, 3H, CH,), 1.49 (t, 3H, CH,), 4.09 (s, 2H, S-CH,), 4.15454 (m, 4H, CH,), 7.20-7.64 (m, 4H, Ar-H), 8.20 (q, 2H, Ar-H), 8.95 (d, lH, Ar-H) v v
(C=O) = 1670 cm-l, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-t DMSO-d, 6 (ppm) = 1.25 (t, 3H, CH,), 4.35 (q, 2H, CH& 7.02-7.56 (m, 4H, Ar-H), 7.83-8.1 (m, 2H, Ar-H), 8.53 (s, lH, Ar-H)
(C=O) = 1680 cm-‘, v (CZN) = 2250 cm-t, (N-H) = 3400 cm-t
(C=O) = 1680 cm-t, v (C=N) = 2240 cm-t, (N-H) = 3400 cm-t DMSO-d, 6 (ppm) = 1.33 (t, 3H, CH,), 4.38 (q, 2H, CH,), 5.1-5.4 (m, 7H, S-CH,)- Q) , 7.19-7.88 (m, 4H, Ar-H), 7.88-8.35 (m, 2H, Ar-H), 8.58 (d, lH, Ar-H) v v
= 1660 cm-l, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-t CDCl, + DMSO-d, 6 (ppm) = 1.42 (t, 3H, CH,), 4.28 (q, 2H, CH,), 4.53 (s, 2H, S-CH,), 7.20-7.50 (m, 9H, Ar-H), 8.OCL8.30 (m, 2H, Ar-H), 8.81 (d, lH, Ar-H)
= 1680 cm-l, v (C=N) = 2250 cm-t, (N-H) = 3400 cm-t CDCl, + DMSO-d, 6 (ppm) = 1.42 (t, 3H, CH,), 4.38 (q. 2H, CH,), 4.49 (s, 2H, S-CH,), 7.20-7.60 (m, 8H, Ar-H), 7.96-8.30 (m, 2H, Ar-H), 8.78 (d, lH, Ar-H)
(C=O) = 1670 cm-t, v (C=N) = 2250 cm-t, (EC-H) = 3240 cm-t DMSO-d, 6 (ppm) = 1.31 (t, 3H, CH,), 3.70 (d, lH, CH), 3.80 (d, H, S-CH,) 4.45 (q, 2H, CH,), 7.08-7.88 (m, 4H, Ar-H), 8.18 (q, 2H, Ar-H), 8.94 (d, lH, Ar-H)
If: 0.5% of theoretical
cl d) ~1 II g) h) I) iI
al R=CH3 b) R*C2H5 C) RzCH2CbH5
R~CH2C~CH R= CH2 CH=CH2 R= CH2COOEt R =CH2CONH2 R: CH2CH2OH R = CH2 4 R = CH2 CgH5 R = CH2C&Ct (L-1
X * NO2
R * C6‘45
b) R =C6HLF c) NHR= d)
Fig 1. Preparation
- N> N-
- CH) N--C6t+5
2980 cm-l, v (C=N) = 2250 cm-l, v (C=O) 1650 cm-l. 1H-NMR (CDCI,): 6 (ppm) = 1.24-1.78 (m, 9H, CH3), 3.38 (q, 2H, SCH,), 4.0-4.5 (m, 4H, CH,), 7.15-7.6 (m, 4H, Ar-H), 8.00-8.44 (m, 2H, Ar-H), 8.92 (d, lH, Ar-H).
m/z = 454, IR (KBr): v (C=N) = 2250 cm-l, lH-NMR (CDCl& 6 (ppm) = 1.48 (t, 3H, CH,), 4.4 (q, 2H, CH,), 4.5 (s, 2H, SCH,), 7.22-7.52 (m, 9H, Ar-H), 8.1-8.3 (m, 2H, Ar-H), 8.85 (d, lH, Ar-H).
3-Benzyl-2-benzylthio-6-(N-ethylcarbazol-3-yl)-3,4-dihydro-loxopyrimidine-.5-carbonitrile 3c This was obtained from 1 and benzyl chloride as described above, yield 25%, mp 189°C. Anal (&H,,N,OS (C, H, N), MS: m/z = 526, IR (KBr): v (CsN) = 2255 cm-l, v (C=O) = 1675 cm-r. lH-NMR (CDCI,): 6 (ppm) = 1.45 (t. 3H, CH,), 4.38 (q, 2H, CH,), 4.59 (s, 2H, SCH,), 5.31 (s, 2H, N-CH,), 7.20-7.58 (m, 14H, Ar-H), 8.0-8.4 (m, 2H, Ar-H), 8.88 (d, lH, Ar-H).
2-Benzylthio-6-(N-ethylcarbazol-3-yl)-4-phenylamino-pyrimidine-5-carbonitrile Sa A solution of 4 (0.2 g, 0.44 mol) in acetone (25 ml) was refluxed with aniline (82 mg, 8.8 mmol) for 4 h, excess of acetone was removed under reduced pressure and residue thus obtained was purified on silica column using ethyl acetatehexane as eluent, yield 80 mg (7.14%) mp 182°C. Anal C,,H,,N,S (C, H, N), MS: m/z = 511, IR (KBr): v (C=N) = 2250 cm-r, v (NH) = 3320 cm-f, lH-NMR (CDCl,): 8 (ppm) = 1.45 (t, 3H, CH,), 4.40 (q, 2H, CH,), 4.42 (s, 2H, SCH,), 7.22-7.63 (m, 14H, Ar-H), 8.12-8.26 (m, 2H, Ar-H), 8.84 (d, lH, Ar-H).
2-Benzylthio-4-chloro-6-(N-ethylcarbazol-3-yl)pyrimidine-5carbonitrile 4 A mixture of 2i (1 g, 2.29 mmol) and N,N-dimethyl aniline (1 ml) was refluxed with POCl, (15 ml) for 4 h. Excess of phosphoryl chloride was removed under reduced pressure and the residue was poured onto crushed ice and stirred. The precipitate thus obtained was crystallized from acetone, yield 0.49 g (39.4%), mp 194°C. Anal C,,H&lN,S (C, H, N), MS:
2-Benzylthio-6-(N-ethylcarbazol-3-yl)-4-(4-fluorophenylamino)pyrimidine-S-carbonitrile 5b A mixture of 4 (0.2 g, 0.44 mmol) and 4-fluoroaniline (0.09 g, 0.81 mmol) in 15 ml of acetone was refluxed for 8 h, worked up as described above and purified on silica gel column using
855 ethyl acetate-hexane as eluent, yield 85 mg (7%), mp 187°C. Anal C32H,FN,S (C, H, N), MS : m/z = 529, IR (KBr): v (C=N) = 2250 cm-i, v (NH) = 3320 cm-l, tH-NMR (CDCI,): 6 (ppm) = 1.45 (t, 3H, CH,), 4.36 (q, 2H, CH,), 4.38 (s, 2H, SCH,), 7.2-7.54 (m, 13H, Ar-H), 8.1-8.25 (m, 2H, Ar-H), 8.82 (d, lH, Ar-H). 2-Benzylthio-6-(N-ethylcarbazol-3-yl)-4-(4-methylpiperazino)pyrimidine-5-carbonitrile SC A solution of 4 (0.2 g, 0.44 mmol) and 4-methylpiperazine (0.081 g, 0.88 mmol) in 20 ml of acetone was refluxed for 6 h. Solvent was evaporated and the residue was washed with water, filtered and crystallized from acetone, yield 0.15 g (65%), mp 203°C. Anal C,,H,&S (C, H, N), MS: m/z 518, IR (KBr): v (C=N) = 2250 cm-t, iH-NMR (CDCl,) 6 (ppm) = 1.46 (t, 3H, CH,), 2.36 (s, 3H, N-CH,), 2.56 (t, 4H), 4.02 (t, 4H), 4.37 (q. 2H, CH,), 4.42 (s, 2H, SCH,), 7.24-7.48 (m, 9H, Ar-H), 8.0-8.19 (m, 2H, Ar-H), 8.69 (d, lH, Ar-H). 2-Benzylthio-6-(N-ethylcarbazol-3-yl)-4-(4-phenylpiperazino)pyrimidine-5-carbonitrile Sd This was prepared from 4 (0.2 g, 0.44 mmol) and 4-phenylpiperazine (0.14 g, 0.86 mmol) as described in the preceeding experiment. The crude product was purified by column chromatography using ethyl acetate-hexane (4: 1) as eluent, yield 40 mg (3%), mp 175’C. Anal (&H,,N,S (C, H, N), MS: m/z = 580, II7 (KBr): v (C=N) = 2250 cm-t, tH-NMR (CDCl,): 6 (ppm) = 1.47 (t, 3H, CH,), 3.32 (t, 4H), 4.15 (t, 4H), 4.38 (q, 2H, CH,), 4.46 (s, 2H, SCH,), 6.9-7.5 (m, 14H, Ar-H), 8.02-8.2 (m, 2H, Ar-H), 8.72 (d, lH, Ar-H). 6-Cyano-7-(N-ethylcarbazol-3-yl)-2,3-dihydrothiazolo[3,2-a]pyrimidine-S-one 6a This was synthesized by the reaction of equimolecular quantity of 1 and 1,2-dibromomethane as reported earlier 1101, yield 50%, mp 285°C Anal C,,H,,N,OS (‘C, H, N), MS: ml; 4 372 IR (KBr): v (C=O) = 1690 cm-t. v (C=N) = 2250 cm-i. tH-NMR (TFA): s’(ppm) = 1.21 (t, 3H, CI$), 3.68 (t, 2H: SCH,), 4.15 (q, 2H, NCH&, 4.58 (t, 2H, NCH,), 7.05-7.35 (m, 4H, Ar-H), 7.58-7.85 (m, 2H, Ar-H), 8.3 (s, lk, Ar-H). 7-Cyano-S-(N-ethylcarbazol-3-yl)-2,3,4-trihydropyrimido[2,1 -b]thiazine-6-one 6b This was obtained by the reaction of 1 with 1,3-dibromopropane as described in the preceeding experiment in 42% yield, mp 235°C. Anal CZ2H,sN,0S (C, H, N), MS: m/z = 386, IR (KBr): v (C=O) = 1690 cm-i, v (C=N) = 2250 cm-i, ‘H-NMR (CDCl, + DMSO-$): 6 (ppm) = 1.45 (t, 3H, CH,), 2.24-2.48 (m, 2H, CH,), 3.29 (t, 2H, SCH,), 4.15 (t, 2H, N-CH,), 7.28-7.6 (m, 4H, Ar-H), 8.1-8.28 (m, 2H, Ar-H), 8.84 (d, lH, Ar-H). 3-Cyano-4-(N-ethylcarbazol-3-yl)-7-nitropyrimido[2,1 -b]benzothiazol-2-one 7a A mixture of 1 (0.5 g, 1.2 mmol), 2,4-dinitrochlorobenzene (0.29 g, 1.43 mmol) and potassium carbonate (0.42 g) in DMF
(15 ml) was stirred for 5 h at room temperature. The reaction content was diluted with water, stirred for 5 min and filtered. The precipitate thus obtained was washed several times with acetone to remove unreacted reactants. The crude product was crystallized from DMF, yield 0.14 g (21%), mp > 300°C. Anal C25H15N503S (C, H, N), MS: m/z = 465, IR (KBr): v (C=O) = 1700 cm-i, v (C=N) = 2250 cm-i. 3-Cyano-7,9-dinitro-4-(N-ethylcarbazol-3-yl)pyrimido[2,1 -b]benzothiazol-2-one 7b This was prepared from the reaction of 1 and picryl chloride in equimolar quantity and isolated as described in the preceding experiment, yield 26%, mp > 300°C. Anal (&H,,N,O,S (C, H, N). MS: m/z = 510, IR (KBr): v (C=O) = 1705 cm-i, v (C=N) = 2250 cm-i. 3,4-Dihydro-6-(N-ethylcarbazol-3-yl)-2-hydrazino-3-methyl-4oxopyrimidine-5-carbonitrile 8 A suspension of 3a (1 g, 2.67 mmol) and hydrazine hydrate (5 ml, 100 mmol) was stirred in methanol (50 ml) at room temperature for 24 h. The reaction mixture was filtered and the filtrate was reduced to a small volume. The precipitate thus obtained was filtered, washed with water and finally crystallized from DMF-ether. vield 0.4 e (42%). mo 279°C. Anal C,H,,N,O (C, H, N), MS: m/z 355,‘ IR (KB;): v (C=O) = 1700 cm-l, v (C=N) = 2250 cm-l, v (NH) = 3400 cm-t, iH-NMR (DMSO-D,): 6 (ppm) = 1.32 (t, 3H, CH,), 3.34 (s, 3H, N-CH,), 4.45 (q, 2H, CH,), 7.1-7.75 (m, 4H, Ar-H), 7.90-8.22 (m, 2H, Ar-H), 8.65 (d, lH, Ar-H).
Acknowledgments The authors thank RSIC, CDRI, Lucknow for providing elemental and spectroscopic analyses and also thank RC Bhagat for technical assistance.
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