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JAC Advance Access originally published online on April 4, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1134-1138; doi:10.1093/jac/dkl095
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Synthesis and antileprosy activity of some dialkyldithiocarbamates

Vadim Makarov1, Olga B. Riabova1, Anatoly Yuschenko2, Nailya Urlyapova2, Adilya Daudova2, Peter F. Zipfel3 and Ute Möllmann3,*

1 Department of Medicinal Chemistry, Research Center for Antibiotics 117105 Moscow, Russia 2 Leprosy Research Institute, 414000 Astrakhan Russia 3 Leibniz Institute for Natural Product Research and Infection Biology—Hans-Knoell Institute D-07745 Jena, Germany

*Corresponding author. Tel: +49-3641-656656; Fax: +49-3641-656660; E-mail: ute.moellmann{at}hki-jena.de

Received 11 January 2006; returned 10 February 2006; revised 24 February 2006; accepted 1 March 2006


   Abstract
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Objectives: To investigate the antileprosy potential of a set of original compounds with antimycobacterial activity.

Methods: We developed a facile synthesis of 2-chloro-3-cyano-5-nitropyridine and synthesized a series of 3-cyano-2-dialkyldithiocarbamoyl-5-nitropyridine derivatives. In vivo therapeutic efficacy against Mycobacterium leprae was assessed in the infected mouse footpad model.

Results: The compounds were active in vitro against Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium vaccae and Mycobacterium fortuitum, with MICs generally in the range of 0.4–6.25 mg/L. Reduction of the bacterial load in vivo in the mouse footpad and toxic side effects were dependent on the individual structure of the compounds and on the doses applied. Compounds 2a, 3a and 3b reduced the number of M. leprae by two orders of magnitude, comparable to the effect of dapsone. Co-administration of compounds 2a and 3a with dapsone synergistically enhanced the activity. In addition, these compounds were well tolerated over the treatment period of 7.5 months.

Conclusions: Individual synthetic dithiocarbamate derivatives have promising antileprosy activity.

Keywords: leprosy , mouse footpad model , antileprosy agents , dithiocarbamates , nitropyridines


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Since the beginning of the 1980s, treatment of leprosy patients has been principally based on two WHO-recommended schemes of multi-drug therapy (MDT) including dapsone, rifampicin and clofazimine. This MDT protocol does consider the specific type of the disease (multibacillary and paucibacillary leprosy), and this type of treatment was implemented because of the development of Mycobacterium leprae resistance to the principal antileprosy drugs (sulphone derivatives) that have been widely used since 1940.1,2 Rifampicin does have a high bactericidal activity against M. leprae, but its long-term use alone might cause drug resistance in M. leprae as well.3 Monotherapy with clofazimine has not been successful.4 The WHO approach is based on the anticipation that a simultaneous administration of two or three bactericidal agents with different chemical structures and mechanisms of action should minimize the development of M. leprae resistance to any single component of MDT, improving antileprosy therapy, reducing the time required for treatment and decreasing the risk of relapse.

Dapsone is the main antileprosy drug because of its inherent level of bactericidal activity, facile low cost production, easy application and minimal side effects. For treatment of multibacillary forms of leprosy, clofazimine is included in WHO-MDT schemes, because of its significant antibacterial and anti-inflammatory activity that reduces leprosy peripheral nerve damage. However, the drug is frequently rejected by patients because it accumulates in the skin and eyes and induces hyperpigmentation.5

A number of compounds, such as minocycline, various fluoroquinolones and macrolides, have high bactericidal activity against M. leprae in mouse footpads. In limited clinical trials a new regimen, comprising rifampicin, ofloxacin and minocycline, has shown particular promise for the treatment of single lesion paucibacillary leprosy.6 However, more time is required for the assessment of these effects since relapses of leprosy are observed up to 10 years after discontinuation of therapy.

In spite of the progress, treatment of leprosy requires regular continued therapy. Poor compliance might contribute to the development of multi-drug resistance. MDT results in a high risk of adverse side effects, including problems related to intolerance of the components. According to recent data,7 the incidence of relapse might increase. There is evidence that the relapse rate registered after MDT completion might be higher than that currently registered.8,9 However, the main shortcoming of the current antileprosy therapy is its long duration. Thus, an urgent task of leprology is the improvement of antileprosy treatment through the reduction of the terms of therapy and the development of more effective supervised MDT regimens that might be administered once a month.

One approach to improve treatment of leprosy patients is the development of new agents with antileprosy activity having modes of action other than those of currently available drugs. Such novel therapeutic agents might be administered alone or in combination with established antileprosy drugs, and consequently improved MDT.

In the course of our programme to develop new antituberculosis drugs using structure–activity relationship (SAR) studies we selected a set of original compounds10,11 with antimycobacterial activity in vitro for the investigation of their antileprosy potential.


   Materials and methods
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Chemistry

Melting points were determined according to the BP procedure and are uncorrected (Electrothermal 9001, GB). If analyses are indicated only by the symbols of the elements, analytical results are within ±0.3% of the theoretical values (Carlo-Erba 5500, Italy). NMR spectra were determined using a Varian Unity Plus 400 spectrometer (USA). Shifts for 1H NMR are reported in ppm downfield from TMS ({delta}). Mass spectra were obtained using a Finnigan SSQ-700 (USA) instrument with direct inject. Silicagel 60 F254 aluminium sheets (Merck Co, Germany) were used for all analytical thin layer chromatography (TLC) experiments. All chemicals were purchased from Lancaster Synthesis (Lancashire, UK).

Synthesis of 2-chloro-5-nitronicotinoyl chloride was performed as described previously.11

2-Chloro-5-nitronicotinamide. To a stirred 50 mL solution of 25% aqueous ammonia was added drop-wise a solution of 5 g (23.0 mmol) of 2-chloro-5-nitronicotinoyl chloride in acetonitrile (10 mL) at –20°C; 10 min later, 50 mL of ethyl acetate was added. The organic phase was separated, washed twice with water, dried over Na2SO4, filtered and concentrated. The crude product was purified by crystallization from ethanol. The yield was 3.8 g (83%). Rf (chloroform–methanol, 9/1) - 0.85. mp 181–183°C. MS m/z 201 (M+). 1H NMR (DMSO-d6) {delta} 9.33 (1H, d, J = 1.9 Hz, CH), 8.59 (1H, d, J = 1.9 Hz, CH), 5.73 (2H, broad c, NH2) ppm. Anal. C6H4ClN3O3, C,H,N.

2-Chloro-3-cyano-5-nitropyridine. A total of 2 g (9.9 mmol) of 2-chloro-5-nitronicotinamide was dissolved in POCl3 (20 mL) and refluxed for 1.5 h, then cooled to 0°C (ice bath), poured into crushed ice, extracted with ethyl acetate, washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The yield was 1.2 g (66%) after recrystallization from aqueous ethanol (75%). mp 121–122°C (lit. 120–122°C12).

4-Alkyldithiocarbamoyl-3-cyano-5-nitropyridines (2a–d). A total of 1.5 g (8.17 mmol) of 2-chloro-3-cyano-5-nitropyridine 1 was dissolved in a mixture of 15 mL of ethyl acetate and 15 mL of ethanol. The reaction mixture was treated with 10 mmol of dialkyldithiocarbamate sodium salt and stored for 2 h at room temperature. The mixture was then poured into 50 mL of cooled water and the resulting yellow precipitate was filtered off. The pure final product was obtained after recrystallization (twice) from ethanol.

3-Cyano-2-diethyldithiocarbamoyl-5-nitropyridine (2a). Yield 73%. Rf (hexane–acetone, 2/1) - 0.35, mp 138–140°C. MS m/z 296 (M+). 1H NMR (DMSO-d6) {delta} 9.58 (1H, d, J = 2.3 Hz, CH), 9.31 (1H, d, J = 2.3 Hz, CH), 3.96 [4H, m, N(CH2)2], 1.38 (3H, t, CH3), 1.21 (3H, t, CH3) ppm. Anal. C11H12N4O2S2, C,H,N,S.

3-Cyano-5-nitro-2-tetramethylendithiocarbamoylpyridine (2b). Yield 81%. Rf (hexane–acetone, 2/1) - 0.30. mp 163–165°C. MS m/z 294 (M+). 1H NMR (DMSO-d6) {delta} 9.56 (1H, d, J = 2.3 Hz, CH), 9.31 (1H, d, J = 2.3 Hz, CH), 3.84 [4H, broad c, N(CH2)2], 2.11–1.89 (4H, m, CH2CH2) ppm. Anal. C11H10N4O2S2, C,H,N,S.

3-Cyano-5-nitro-2-pentamethylendithiocarbamoylpyridine (2c). Yield 54%. Rf (hexane–acetone, 2/1) - 0.50. mp 171–173°C. MS m/z 308 (M+). 1H NMR (DMSO-d6) {delta} 9.55 (1H, d, J = 2.3 Hz, CH), 9.31 (1H, d, J = 2.3 Hz, CH), 3.76 [4H, m, N(CH2)2], 2.11–2.08 [6H, m, (CH2)3] ppm. Anal. C12H12N4O2S2, C,H,N,S.

3-Cyano-2-hexamethylendithiocarbamoyl-5-nitropyridine (2d). Yield 89%. Rf (hexane–acetone, 2/1) - 0.45. mp 168–170°C. MS m/z 322 (M+). 1H NMR (DMSO-d6) {delta} 9.58 (1H, d, J = 2.3 Hz, CH), 9.33 (1H, d, J = 2.3 Hz, CH), 3.61 (2H, m, NCH2), 3.42 (2H, m, NCH2), 1.64–1.98 [8H, m, (CH2)4] ppm. Anal. C13H14N4O2S2, C,H,N,S.

Synthesis of 6-methoxy(dimethylamino)-5-nitro-4-tetrahydromethylendithio-carbamoyl-pyrimidines 3a and 3b and 3,5-dinitro-2-tetrahydromethylendithio-carbamoylbenzamide 4 was performed as described previously.10

In vitro assay of antibacterial activity

Microorganisms. Mycobacterium smegmatis HKI 056 (DSM 44200), Mycobacterium vaccae IMET 10 670, Mycobacterium aurum SB66 and Mycobacterium fortuitum B were obtained from the culture stock of the Hans-Knoell Institute. The strains were grown for 2 days under shaking at 37°C in a medium containing 10 g of glycerol, 5 g of meat extract, 5 g of pancreatic-digested casein peptone and 3 g of NaCl in 1 L of distilled water.

MICs. The antibacterial activity of the compounds was studied by the determination of MICs according to the NCCLS guidelines13 using a broth microdilution method in Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) with a final inoculum of 5 x 105 cfu/mL. Microtitre plates were read after incubation at 37°C for 24 h with a Nepheloscan Ascent 1.4 automatic plate reader (Labsystems, Vantaa, Finland). The MIC value represents the lowest dilution of the compound in which no bacterial growth was detected.

In vivo assay of antileprosy activity in the mouse footpad model

Animals. A total of 320 specific-pathogen-free CBA mice (8 weeks old, 18–22 g) were obtained from the animal house of the Leprosy Research Institute, Astrakhan.

Microorganism and inoculation. A strain of M. leprae was previously isolated from a leprosy patient. Mice were infected in the right hind footpad with M. leprae using a dose of 5 x 103 microorganisms according to the method of Shepard,14 recommended by the WHO for antileprosy drug screening.15 At the end of the experiment the tissues of paw cushions of mice were carefully slashed and crushed. This material was suspended in 2.0 mL of distilled water containing 0.1% albumin. The suspension was centrifuged for 3 min at 1000 rpm to separate large tissue particles, and the supernatant was used for the preparation of smears. For this purpose, 10 µL of M. leprae suspension was diluted (v/v) with 0.1% albumin in water and spread in an area of 1 cm2. The smears were dried, fixed and stained using the Ziehl–Neelsen method.

The number of mycobacteria in 1.0 mL of suspension was determined microscopically using the method of Shepard and McRae.16 The total number of the sight fields calculated for each suspension was 60. The total number of mycobacteria in the suspension was calculated using the following formula:

Formula
where R1 is the radius of the smear area and R2 is the radius of the sight field of the microscope measured in micrometres. After that the suspension was diluted to achieve the inoculum required, 5 x 103 M. leprae cells/0.03 mL, which was injected into the paw of one mouse.

The difference in the number of M. leprae between the control animals and the animals of the groups that received test compounds allows us to assess the compounds' ability to inhibit growth of leprosy bacilli.

Treatment of mice. For the in vivo trials, 323 mice were clustered into 17 experimental groups: 15 groups to assay the antileprous activity of selected compounds and 2 control groups, one as an infection control and the other as a dapsone therapeutical control. Starting the day after inoculation, the compounds under study were administered in daily doses of 10, 20 and 30 mg/kg (19 mice per group) orally, five times a week. Before administration, compounds were mixed with starch gel and 0.5 mL of the suspensions was administered to the animals orally by gavage. The doses were selected according to the doses we used for analogue compounds10 in the murine tuberculosis model. All animals were sacrificed 7.5 months following infection and the number of M. leprae in the footpads was determined.

Animal experiments were performed according to international rules and the Russian laws for animal welfare and were approved by the Ministry of Health of the Russian Federation, Licence No. 1243/04.

Statistical analysis. Differences in the efficacy of the treatment between the groups, expressed by the number of M. leprae, were determined using the Student's t-test. P < 0.05 indicated statistical significance.


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Synthesis

Through our efforts related to the search for new antimicrobial compounds we observed that ortho nitro-dialkyldithiocarbamate derivatives have potent antimycobacterial activity.10 As a result of different chemical modifications of this basic structure together with SAR studies we replaced the electron accepting nitro-group with a cyano group, which has similar electron affinity. We developed a facile synthesis of 2-chloro-3-cyano-5-nitropyridine 1 as a basic compound for future synthesis. The previous synthetic route17 was based on bromination of furan-2-carboxylic acid to induce transformation to 2,3-dibromo-4-oxo-cis-crotonic acid followed by condensation with cyanoacetamide. This process is not appropriate for the synthesis of bulk quantities of 1. As a viable alternative, we started with 2-hydroxynicotinic acid, which was consecutively nitrated, transformed to the corresponding chloroanhydride by treatment with thionyl chloride and reacted with aqueous ammonia, producing the corresponding amide. The amide was then treated with POCl3 to give the final 2-chloro-3-cyano-5-nitropyridine 1. The chlorine substituent is highly reactive and susceptible to nucleophile replacement as appropriate for the preparation of dialkyldithiocarbamate derivatives 2a–d (Figure 1). The structures of compounds 3a, 3b and 4, which were synthesized as described previously,10 are presented in Figure 2.


Figure 1
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  		Figure 1. Schematic representation of 3-cyano-2-dialkyldithiocarbamoyl-5-nitropyridine 2a–d synthesis.

 

Figure 2
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  		Figure 2. Structures of 5-nitro-4-tetrahydromethylendithiocarbamoyl-6-R-pyrimidines3a and 3b and 3,5-dinitro-2-tetrahydromethylendithiocarbamoyl-benzamide4.

 
In vitro antibacterial activity

All compounds tested were active against fast growing mycobacteria, particularly against M. vaccae (Table 1). The MICs of the seven tested compounds for M. smegmatis, M. aurum, M. vaccae and M. fortuitum ranged from 0.4 to 6.25 mg/L (except the MIC of compound 4 for M. smegmatis). For compounds 2a and 3a, which subsequently did not exhibit toxicity towards the mice, the MICs ranged between 0.4 and 3.12 mg/L and between 0.8 and 3.12 mg/L, respectively


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  		Table 1. Activity of ortho nitro-dialkyldithiocarbamate derivatives and of ciprofloxacin (CIP) as reference compound against fast growing mycobacteria

 
In vivo therapeutic effects in the mouse footpad model

After 3 weeks, fatalities were noted. At the end of the third week, two mice in groups 6 and 8 perished and subsequently animals in other groups died. Therapy was discontinued 26 days after the experiments had started. However, fatalities were noted through day 31 in groups 3–8 and 12–15 (Table 2). In contrast, all animals in the control group and in the groups treated with compounds 2a, 3a and dapsone alone or in combination survived (Table 2). After day 32 and through the next month, all animals survived. Because the main objective of the experiment was to determine a correlation between structure and activity and to identify the most active compound, the treatment was continued starting from day 56 with 5-fold reduced doses (Table 3). After this, neither acute nor chronic toxic effects were observed. Animals were active and showed normal feeding and normal social behaviour. At the end of the experiment the weight of the animals in all groups was 29–32 g. Thus, the deaths of animals in the first phase of the experiment might be explained by the toxic side effects of compounds 2b, 2c, 2d and 4 at higher dosage.


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  		Table 2. Initial dosage of treatment groups and number of animal deaths after 31 days of treatment

 

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  		Table 3. Dosage of treatment groups starting from day 56 after infection, and results of M. leprae count in footpads

 
After 7.5 months the number of leprosy bacilli in the footpads of the infected animals in control groups approached (2.71 ± 0.49) x 107. In correlation to the data reported in the literature18,19 in the group treated with dapsone, the count of M. leprae was significantly reduced to (0.030 ± 0.005) x 107. In the group that received compound 2a, the total count of mycobacteria (0.042 ± 0.014) x 107 was significantly lower than that in the untreated control group, but slightly higher than that in the group given dapsone. Co-administration of compound 2a with dapsone resulted in a synergistic antibacterial effect on M. leprae. In this group the number of mycobacteria in footpads of mice was (0.021 ± 0.003) x 107, i.e. lower than that in animals treated with dapsone alone and two times lower than that in mice given pyridine 2a alone.

Compound 3b showed antileprosy activity at a dose of 6 mg/kg, which is comparable to that of dapsone at 2 mg/kg. The residual number of bacteria was (0.029 ± 0.006) x 107. At a dose of 2 mg/kg, compound 3b reduced bacterial counts to (0.070 ± 0.020) x 107.

The number of M. leprae in footpads of mice given compound 3a at a dose of 2 mg/kg was (0.660 ± 0.120) x 107, i.e. significantly lower than the control but higher compared with dapsone. Increase of the dose to 6 mg/kg resulted in a pronounced antileprosy effect; the count of mycobacteria in footpads of mice of the corresponding group was (0.020 ± 0.006) x 107, i.e. less than in dapsone-treated mice. Combination of compound 3a at a dose of 4 mg/kg with dapsone at a dose of 2 mg/kg resulted in an additional reduction of M. leprae count [(0.017 ± 0.002) x 107] that was significantly lower than that in animals given only dapsone (Table 3).

The M. leprae counts in footpads of mice treated with compounds 2b, 2c, 2d and 4 were about one order of magnitude lower than that in the control group mice but also about one order of magnitude higher than that in dapsone-treated mice (Figure 3).


Figure 3
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  		Figure 3. Antileprosy activity of new dialkylditiocarbamate derivatives in the mouse footpad model.

 
In conclusion, all synthetic dithiocarbamate derivatives studied here inhibit growth of M. leprae in mouse footpads. Antileprosy activity and toxic side effects are variable and dose-dependent. Compounds 2a, 3a and 3b are most promising as potential antileprosy agents due to both the strong reduction in M. leprae counts and the good tolerability.


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None to declare.


   Acknowledgements
 
We gratefully acknowledge the financial support from the BMBF (FKZ 0311552) and from Medac GmbH, Hamburg, Germany and of the Federal Agency for Science and Innovations of the Russian Federation (Contract No. 1/05).


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1 Faget GH, Pogge RC, Johansen FA, et al. (1943) La prominoterapia de la lepra. Estudios en Progresso. Int J Lepr 11:52–63.

2 Lowe J. (1950) Treatment of leprosy with diamino-diphenyl sulphone by mouth. Lancet i:145–50.

3 Jacobson RR and Hastings RC. (1976) Rifampin-resistant leprosy. Lancet ii:1304–5.

4 Yawalkar SJ and Vischer W. (1979) Lamprene (clofazimine) in leprosy. Lepr Rev 50:135–44.[Medline]

5 Anon. Clofazimine: new products. Major antileprous drug. (1999) Prescrire Int 40:44–6.

6 Ji B, Sow S, Perani E, et al. (1998) Bactericidal activity of a single-dose combination of ofloxacin plus minocycline, with or without rifampin, against Mycobacterium leprae in mice and in lepromatous patients. Antimicrob Agents Chemother 42:1115–20.[Abstract/Free Full Text]

7 Bakker MI, Hatta M, Kwenang A, et al. (2005) Prevention of leprosy using rifampicin as chemoprophylaxis. Am J Trop Med Hyg 72:443–8.[Abstract/Free Full Text]

8 You EY, Kang TJ, Kim SK. (2005) Mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea. J Infect 50:6–11.[CrossRef][Medline]

9 Zhang L, Namisato M, Matsuoka M. (2004) A mutation at codon 516 in the rpoB gene of Mycobacterium leprae confers resistance to rifampin. Int J Lepr Other Mycobact Dis 72:468–72.[Medline]

10 Makarov V and Möllmann U. Dithiocarbamate derivatives and their use as antibacterial agents. PCT Int. Appl. WO 03/042186.

11 Makarov V, Möllmann U, Ryabova O, et al. 2-Dithiocarbamatopyridin-derivate, Verfahren zu ihrer Herstellung und ihre Verwendung. German Appl. 102005031353.1; Priority 01.07.2005.

12 Fanta PE and Stein RA. (1955) The condensation of sodium nitromalonaldehyde with cyanoacetamide. J Amer Chem Soc 77:1045–6.[CrossRef]

13 National Committee for Clinical Laboratory Standards. (1997) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Fourth Edition: Approved Standard M7-A4 (NCCLS, Villanova, PA, USA).

14 Shepard CC. (1960) The experimental disease that follows the infection of human leprosy bacilli info footpads of mice. J Exp Med 112:445–54.[Abstract]

15 Anon. WHO expert committee on leprosy. (1977) World Health Organ Tech Rep Ser 607:7–48.

16 Shepard CC and McRae DH. (1968) A method of counting acid-fast bacteria. Int J Lepr Other Mycobact Dis 36:78–82.[Medline]

17 Winn M, Zydowsky TM, Altenbach RJ. (1993) 2-(Alkylamino)nicotinic acid and analogs. Potent angiotensin II antagonists. J Med Chem 36:2676–88.[Medline]

18 Gidoh M and Tsutsumi S. (1989) The inhibitory effects by combined doses of DDS and several immunostimulants on the growth of leprosy bacilli inoculated into footpads of hybrid nude mice, Jcl:AF-nu. Nippon Rai Gakkai Zasshi 58:241–9.[Medline]

19 Gelber RH. (1998) Another view of the therapy of leprosy. Antimicrob Agents Chemother 42:3334–6.[Free Full Text]


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