Suitably functionalised pyrimidines as potential antimycotic agents

Article abstract of DOI:10.1016/S0960-894X(00)00091-3

Various suitably functionalised pyrimidine derivatives have been synthesized to explore their potential as antimycotic agents. Some of the synthesized compounds 4c, 4d, 8a-e have shown highly significant in vitro antifungal activity against five human pathogenic fungi. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00091-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 703±706
Suitably Functionalised Pyrimidines as Potential Antimycotic
Agentsy
Nidhi Agarwal, a Sandeep K. Raghuwanshi, b D. N. Upadhyay, a P. K. Shukla b
and Vishnu J. Ram a,*
aDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow, 226001, India
bDivision of Medical Mycology, Central Drug Research Institute, Lucknow, 226001, India
Received 22 December 1999; accepted 22 January 2000
AbstractÐVarious suitably functionalised pyrimidine derivatives have been synthesized to explore their potential as antimycotic
agents. Some of the synthesized compounds 4c, 4d, 8a±e have shown highly signi®cant in vitro antifungal activity against ®ve
human pathogenic fungi. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
This paper reports on the synthesis of various pyr-
imidine derivatives with di€erent substituents across the
ring skeleton to explore their therapeutic potential as
antifungal agents. Among all the synthesized 2,4,6-
trisubstituted pyrimidine-5-carbonitriles, 4-chloro-2,6-
disubstituted pyrimidine-5-carbonitriles are found sig-
ni®cantly active against ®ve human pathgoenic fungi,
Aspergillus fumigatus (Af), Candida albicans (Ca),
Cryptococcus neoformans (Cn), Trichophyton menta-
grophytes (Tm) and Sporothrix schenckii (Ss) in in vitro
screen. The in vitro screening of all the synthesized
compounds was determined by 2-fold serial dilution
technique. The activity of these compounds was expres-
sed in terms of minimum inhibitory concentration
(MIC), considering ketoconazole as a standard drug.
The most potent compound amongst all the screened
compounds was 4d which displayed signi®cant activity
against Af, Tm and Cn at 0.002, 0.005 and 1.5 mg/ml
concentration respectively. In general 4-chloro-2,6-di-
substituted pyrimidines 4a±e and 8a±d were found highly
active against Af and Tm. Replacement of 4-chloro
group by nucleophilic substituents such as NHR, and
SH group, a complete loss of activity pro®le of all the
synthesized compounds is presented in the Table 1.
The incidence of invasive fungal infections has largely
been attributed to the rising number of immunocom-
promised patients. The commonest cause of invasive
fungal infections in human are the species of Aspergillus
followed by Candida and Cryptococcus neoformans. The
super®cial fungal infections, very common in major
populations is caused by species of Trichophyton men-
tagrophytes, T. rubrum and Sporothrix schenckii. Because
of a wide prevelence of mycoses due to soil borne or
airborne transmission of the systemic fungal pathogens
and lack of broad spectrum antifungals led to the search
for newer prototype molecules to exhibit highly sig-
ni®cant antimycotic activity.
Among various nitrogen heterocycles, derivatives of
azole such as imidazole and triazole are proved to be
clinically potent and useful antifungal agents.1 7 Pyr-
imidine, an enlarged ring size of imidazole by one car-
bon atom is not extensively exploited as antimycotic
agents. Except for a few pyrimidine derivatives such as
trimethoprim and 5-¯uorocytosine8 none of the other
compounds are found clinically useful. Only 5-¯uoro-
cytosine alone is in clinical use against systemic fungal
infections.The antifungal activity of 5-¯uorocytosine is
due to its inhibitory property of a signi®cant enzyme
thymidylate synthase, responsible for DNA synthesis.8
Synthesis
Various pyrimidine derivatives of the prototypes 4, 8, 9
and 10 have been synthesized9 to explore their anti-
fungal activity. 4-Chloro-6-methylthio-2H/substituted
pyrimidine-5-carbonitrile (4a±e) have been synthesized
by two di€erent routes. In both the routes appropriate
*Corresponding author. Tel.: +91-522-212411 ext. 18; fax: +91-522-
2223405.
yC.D.R.I. Communication No. 6013.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00091-3

704
N. Agarwal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 703±706
Table 1. In vitro antifungal activity of pyrimidine derivatives 4a±e
and 8a±f, 9 and 10 against human pathogenic fungi
The other prototype of chloropyrimidines (8a±f) were
prepared starting from 6-aryl-5-cyano-2-thiouracil9 (6).
Regioselective alkylation of 6 with methyl iodide, led to
the formation of 6-aryl-2-methylthio-4-oxo-3,4-dihydro-
5-carbonitrile12 and were halogenated by phosporyl
chloride to yield 6-aryl-4-chloro-2-methylthiopyr-
imidine-5-carbonitrile. The halo pyrimidines are highly
activated due to vicinal presence of electron with-
drawing CN substituent and are prone to yield 6-aryl-
Compound no.
Minimum inhibiting concentration (MIC)
in mg/ml
Af
Tm
Ca
Cn
Ss
4a
4b
4b
4d
4e
8a
8b
8c
8d
8e
8f
9a
9b
9c
0.39
6.25
0.02
0.002
0.78
0.09
1.56
0.04
0.09
0.04
50
>100
>100
>100
>100
0.7
0.39
1.56
12.5
0.005
0.19
0.09
0.39
0.09
0.39
0.19
50
>100
>100
>100
>100
0.1
25
25
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.3
1.56
1.56
>100
1.56
6.25
50
6.25
12.5
>100
50
50
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
25
4-arylamino-2-methylthio-5-carbonitriles (9a±c).12
A
reaction with thiourea provided corresponding 6-(3-
¯uorophenyl-4-mercapto-3-methylthio-5-carbonitrile (10)
Scheme 2.
3.12
50
1.56
>100
>100
>100
>100
>100
>100
0.1
Biological Activity
The in vitro antifungal activity was evaluated by 2-fold
serial dilution technique13 against ®ve human patho-
genic fungi viz. A. fumigatus, C. albicans, C. neoformans,
S. schenckii and T. mentagrophytes. Sabouraud's dex-
trose agar (SDA) slant growth of the pathogens for
24±48 h (yeasts) or 7 days (mycelial fungi) were sus-
pended in Sabouraud's dextrose broth (SDB). The col-
ony forming units (cfu) of the seeded broth were
estimated by dilution and plating technique and adjus-
ted in the range of 104±105 cfu/ml.
10
Ketoconazole
ketene dithioacetals are used as precursor. A reaction
of ethyl 2-cyano-3,3-dimethylthioacrylate10 (1a) with
amidine in alcohol at re¯ux temperature yielded 6-
methylthio-4-oxo-2H/substituted-3,4-dihydropyrimidines
(3a±e) which were also obtained11 from the reaction of
2-cyano-3,3-dimethylthioacrylamide and acid anhy-
dride. The oxopyrimidines (3a±e) on halogenation with
phosphoryl chloride provided chloropyrimidines (4a±e),
Scheme 1.
The test compounds were dissolved in DMSO to get a
stock solution of 1 mg/ml. To 1.8 ml seeded broth was
added 0.1 ml of the stock test solution. One milliliter of
Scheme 1.

N. Agarwal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 703±706
705
Scheme 2. Reagents/conditions: (i) EtOH/100 ꢀC; (ii) POCl3/re¯ux; (iii) (RCO)2O/re¯ux; (iv) RX/Na2CO3/DMF/0 ꢀC; (v) arylamine/EtOH/100 ꢀC;
(vi) thiourea/EtOH/re¯ux.
this solution was further diluted with 1 ml of seeded
broth to make second dilution and so on until 16 such
dilutions were obtained. A set of assay tubes with see-
ded broth and solvent were kept as control. The tubes
were incubated at 28‹1 ꢀC and observed for MIC after
24±96 h depending upon the time required for optimum
growth of the test organism. Ketoconazole was used as
the standard drug.
in¯uence MIC of the test compounds. Halophenyl sub-
stituent at position 6 in 8a±f ameliorates the activity
while the phenyl group with electron donating sub-
stituent (8f), attenuates it. Pyrimidines (9a±c, 10) with
nucleophilic substituents (NHAr,SH) at position 4
without any change in other substituents, completely
lost the antifungal activity.
The antifungal activity pro®le of various pyrimidine
derivatives 4a±e, 8a±f, 9 and 10 revealed that 4-chloro
substituent in the pyrimidine skeleton is a must to
express signi®cant activity. Substituents of positions 2
and 6 either ameliorate or attenuate the antimycotic
activity depending upon the nature and size of groups.
As evident from the activity of 4a±e, a mere change in
the size of substituent at position 2, greatly in¯uences
the activity. The most active compound in 4-chloropyr-
imidines (4a±e, 8a±f), 4d is highly active in in vitro
screen against Af and Tm. Except amphotericine B none
of the clinically used antifungals known so far which
have MIC of 0.002 and 0.005 mg/ml concentration in in
vitro screening against Af and Tm. An increase in the
size of the substituent at position 2 from S-CH3 to S-
CH2C6H5 in 4d, attenuated the activity. Similarly in
other 4-chloropyrimidines 8a±b, an increase in the size
of alkyl substituent at position 2 decrease in the activity
was observed. Pyrimidines with or without alkyl or aryl
substituent at position 2 in 4a±c, displayed signi®cant
activity. However, 4-chloro-6-methylthio pyrimidine-5-
carbonitrile (4a) was found highly active against all the
®ve test fungi. Substituents at position 6 in 8a±f also
Acknowledgements
Nidhi Agarwal and Sandeep K. Raghuwanshi are
thankful to ICMR for ®nancial support of the project.
References and Notes
1. Cheng, C. C. Prog. Med. Chem. 1969, 6, 67.
2. (a) Scherkenbeck, J.; Himmler, T.; Beohm, S.; Hagemann,
H.; Stroecl, K.; Dutzmann, S.; Dehne, H. W.; Haenssler, G.
Ger. O€en DE 4,208,050, 23rd Sep. 1993; Chem. Abstr. 1994,
120, 107033m. (b) Walker, K. A. M.; Braemer, A. C.; Hitt, S.;
Jones, R. E.; Mathews, T. R. J. Med. Chem. 1978, 21, 480.
3. Obata, T.; Fujii, K.; Tsutsumiuchi, K.; Nakamoto,Y. Eur.
Pat. Appl. EP 543,402, 26 May 1993; Chem. Abstr. 1994, 120,
107043q.
4. Wachtler, P.; Paulus, W.; Ludwig, Georg-Wilhelm, Eur.
Pat. Appl. EP 559, 002; 8 September 1993; Chem. Abstr. 1994,
120, 107036q.
5. Sakai, S.; Hase, H.; Narita, I.; Hatano, K.; Takunaga, H.
JP05,294,945/93,294,945; Chem. Abstr. 1994, 120, 217725w.
6. Rollas, S.; Kalyoncuoglu, N.; Sur-Altiner, D.; Yegenoglu,
Y. Pharmazie 1993, 48, 308.

706
N. Agarwal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 703±706
7. Kreutzberger, A.; Burger, A. Arch. Pharm. (Weinheim)
1993, 328, 473.
8. Shoichet, B. K.; Stroud, R. M.; Santi, D. U.; Kuntz, I. D.;
Perry, K. M. Science 1993, 259, 1445.
10. Gompper, R.; Top¯, W. Chem. Ber. 1962, 95, 2861.
11. Dornow, A.; Dehmer, K. Chem. Ber. 1967, 100, 2577.
12. (a) Ram, V. J. J. Prakt. Chem. 1989, 331, 893. (b) Ram, V.
J. Arch. Pharm. (Weinheim) 1990, 323, 895.
9. Ram, V. J.; Vanden Berghe, D. A.; Vlietinck, A. J. Hetero-
cyclic Chem. 1984, 21, 1307.
13. Dhar, M. L.; Dhar, M. M.; Dhawan, B. N.; Mehrotra, B.
N.; Ray, C. Indian J. Exp. Biol. 1968, 6, 232.