Journal of
Entomology and Nematology

  • Abbreviation: J. Entomol. Nematol.
  • Language: English
  • ISSN: 2006-9855
  • DOI: 10.5897/JEN
  • Start Year: 2009
  • Published Articles: 135

Full Length Research Paper

Determination of the larvicidal activity of benzoyl thiosemicarbazone and its Ni(II) complex against Aedes aegypti and Anopheles darlingi larvae in Amazonas, Brazil

Rochelly da Silva Mesquita
  • Rochelly da Silva Mesquita
  • Coordenação de Sociedade, Ambiente e Saúde, Instituto Nacional de Pesquisas da Amazônia- INPA, Av. André Araújo, 2936, Aleixo, 69083-000, Manaus – Amazonas, Brazil.
  • Google Scholar
Wanderli Pedro Tadei
  • Wanderli Pedro Tadei
  • Coordenação de Sociedade, Ambiente e Saúde, Instituto Nacional de Pesquisas da Amazônia- INPA, Av. André Araújo, 2936, Aleixo, 69083-000, Manaus – Amazonas, Brazil.
  • Google Scholar
Ana Mena Barreto Bastos
  • Ana Mena Barreto Bastos
  • Instituto Federal de Educação, Ciência e Tecnologia do Amazonas - IFAM, Av. Sete de Setembro, 1975, Centro, 69020-120, Manaus–Amazonas, Brazil.
  • Google Scholar

  •  Received: 17 April 2018
  •  Accepted: 30 August 2018
  •  Published: 31 August 2018


Due to the resistance of some mosquitoes to pyrethroids insecticides, new synthetic compounds are of great interest for the development of new insecticides against vectors of tropical diseases, especially in the Amazon region. Our aim was to synthesize and evaluate the larvicidal potential of benzoyl thiosemicarbazone and its Ni(II) complex against larvae of Aedes aegypti............ and Anopheles darlingi........... . The compounds were synthesized from thiosemicarbazide according to the literature, and the larvicidal potential was evaluated in triplicate at concentrations of 7 to 500 µg/mL. Benzoyl thiosemicarbazone and its Ni(II) complex showed an LC50 of 42.09 and 42.28 µg/mL, respectively, against . Ae. aegypti larvae. For .An. darlingi larvae, the LC50 values of benzoyl thiosemicarbazone (4.77 µg/mL) were lower than its Ni(II) complex (7.33 µg/mL). Benzoyl thiosemicarbazone presented satisfactory results against the larvae, and due to the insecticidal potential of this substance, the development of new chemical insecticides may be possible.


Key words: Benzoyl thiosemicarbazone, nickel (II), larvicidal activity; Aedes aegypti, Anopheles darlingi.




Many pathogens can be transmitted to humans from infected mosquitoes such as Aedes aegypti (Linnaeus, 1762), a species responsible for the transmission of dengue fever, yellow fever, chikungunya Chikungunya and Zika. In Brazil, this vector is one of the main public health problems, since it is extremely urban, has high population growth rates and is difficult to control (Simon et al., 2008; Puccioni-Sohler et al., 2017). Anopheles darlingi (Root, 1926) is of great medical relevance, as it is the vector responsible for the transmission of Malaria in Brazil, where it is basically confined to the Amazon region. Among the anopheline mosquitoes found in the region, A. darlingi is the species that  most  benefits  from   human   modifications   to  the menvironment. It is highly anthropophilic and endophagic (Deane, 1986; Tadei et al., 1998; Maciel-de-Freitas et al., 2012; Sinka et al., 2012). In this case, the control of mosquito populations is performed with insecticides, which, despite numerous records of resistance and high toxicity to nontarget organisms, still provide one of the most effective methods for combatting mosquitoes in endemic areas (Rivero et al., 2010; WHO, 2017).
Thiosemicarbazones belong to the thiourea group, an important class of N and S donor compounds that have high pharmacological potential, and in many cases, the mechanism of action of thiosemicarbazone is associated with complexed transition metals. From a biological point of view, metal complexes are more bioactive than free ligands, particularly thiosemicarbazones, which are active only when complexed with transition metals (Wasi and Singh, 1987; Rosu et al., 2010; Viñuelas-Zahínos et al., 2011; Netalkar et al., 2015).
The pharmacological applications of the different structural derivatives of thiosemicarbazones and their metal complexes include antifungals (Parrilha et al., 2011), cytotoxics (Rebolledo et al., 2005; Braga et al., 2016), antibacterials (Despaigne et al., 2010), antimalarials (Greenbaum et al., 2004; Chellan et al., 2010; Nandal and Deep, 2017) and insecticides (Rayms-Keller et al., 1998; Wang et al., 2010; Silva et al., 2015). Metal complexes or even free metal ions are toxic to aquatic organisms and may be found available at low levels in the environment (Arnold et al., 2005). Thus, due to the high incidence of vector mosquitoes in the region, the permanent and semi-permanent expressions of Aedes and Anopheles mosquitoes in the urban areas of cities and control actions on immature forms represent a functional alternative for the control of insects (WHO, 2014).
The synthesis and biological activity of the 1-benzoyl analogue have been reported in the literature (Xue et al., 2007; Pingaew et al., 2010). In the present study, we examined the larvicidal activity of benzoyl thiosemicarbazone and its nickel (II) complex in bioassays against A. aegypti and A. darlingi, for the control of tropical diseases in the Amazon. Our study addresses the process for obtaining the substance and its nickel complex as well as their larvicidal activity against vectors of dengue and malaria in the Amazon region not yet described in the literature.



Synthesis of Benzoyl thiosemicarbazone (HBzS) and the nickel metal complex [Ni(BzS)2]
All reagents were purchased from the Sigma-Aldrich and used without further purification. The compounds were synthesized (Beraldo et al., 1997 and; Pingaew et al., 2010). The benzoyl thiosemicarbazone was prepared using 11 mmols of thiosemicarbazide for 11 mmol of benzaldeyde. The mixture was heated under reflux for 8 h  and  ethanol  as  a  solvent.  The nickel metal complex was prepared using 2 mmols of Benzoyl thiosemicarbazone for nikel chloride II heated under reflux and drops of amonium hidroxide in ethanol. The FT-IR spectra (KBr) were recorded on Perkin Elmer 283B (4000–400 cm-1) spectrometer. The 1H NMR and 13C NMR were obtained on a Unity Inova 500 Varian spectrometer, em DMSO-d6.
Synthesis of compounds
HBzS: yellow crystals; yield (%): 70; melting point (°C): 205; IR (KBr, cm-1): ν (C=N): 1575; ν (C=S):857; ν (N-H): 3176; ν (N-H2): 3052. 1H NMR (500 MHz, DMSO-d6) (δ): 8.71 (s, 1H), 7.89 – 7.87 (dd, 2H, J: 2 Hz), 7.52 – 7.48 (m, 2H J: 5 Hz), 7.45 – 7.40 (m, 1H, J: 7.5Hz), 6.85 (s, 3H), 3.40 (s, 2H). 13C NMR   (500 MHz, DMSO-d6) (δ):184.14; 161.44; 133.78; 131.37; 128.92; 128.36; 125.76; 76.95.
[Ni(BzS)2]: green solid; yield (%): 65; melting point (°C): > 300; IR (KBr, cm-1): ν (C=N): 1575; ν (C=S): 751; ν (Ni-N): 497; ν (Ni-S): 448; ν (N-H): 2950; ν (N-H2): 3052. 1H NMR (500 MHz, DMSO-d6) (δ): 8.71 (s, 1H), 7.89 - 7.86 (dd, 3H, J: 2 Hz), 7.52 – 7.48 (m, 2H, J: 5Hz), 3.34 (s, 2H). 13C NMR (500 MHz, DMSO-d6) (δ): 162.12; 134.49; 132.05; 129.61; 129.06; 127.98.
The formation of all compounds was confirmed by IR spectroscopy, 1H NMR and 13C NMR. The IR spectrum of Benzoyl thiosemicarbazone showed absorption bands at 3052, 1575 and 857 cm-1, corresponding to the NH, C=N and C=S groups, respectively. The IR spectrum of the nickel metal complex showed absorption bands at 3052-2950, 1625, 751, 497 and 448 cm-1, corresponding to the NH, C=N, C=S, Ni-N and Ni-S groups, respectively. Compounds showed a sharp singlet observed at δ 8.70 and 8.71, which confirmed the presence of the NH to HBzS and [Ni(HBzS)2], H-aromatic rings at δ 7.42 – 7.87 and δ 7.50-7.89, respectively. The 13C NMR spectra of compounds showed peaks at δ 193.24 and δ 161.33, corresponding to C=S carbon to HBzS and [Ni(HBzS)2], respectively. The above values are evident for formation of compounds (Figure 1).
Study site and period
The study was conducted in 2011 in the city of Manaus, Amazonas State, Brazil (-3.096240 latitude, -59.986194 longitude), located in the northern region of the country, which comprises the Brazilian Amazon region. Manaus has a population of 1,802,014 people according to the last census of 2010.
Mosquito collection and maintenance
The larvae were obtained from the insectarium of the Laboratory of Malaria and Dengue, National Institute of Amazonian Research (Instituto Nacional de Pesquisas da Amazônia - INPA), located in Manaus. The larvae remained in trays and were fed with TetraMin® (fish feed); the adult population was kept in cages with cotton soaked in 10% sucrose solution, whereas the females were also fed blood every other day for egg development. Plastic cups with moistened filter paper strips were provided for oviposition by pregnant females. The population was maintained under laboratory conditions of 26 ± 2°C and 70-80% relative humidity.
Larvicidal activity assays
Benzoyl thiosemicarbazone and its Ni(II) complex were synthesized and characterized according to Beraldo et al. (1997) and Pingaew et  al.  (2010).  In  the  assays,  the  compounds  were  dissolved  in dimethyl sulfoxide (DMSO) and evaluated at concentrations of 15.6 to 500 µg/mL against A. aegypti larvae and from 7.8 to 250 g/L against A. darlingi larvae. The assays were performed in triplicate in plastic cups containing 10 mL of distilled water, 10 larvae, feed and 100 μL of the evaluated concentration. Control was performed with DMSO. After 24 and 48 h, the number of dead larvae was recorded, and the median lethal concentration (LC50) was calculated (Dulmage et al., 1990; WHO, 2005).
Statistical analysis
The results were considered acceptable when the control mortality was less than 10%, and the number of dead larvae in the control was adjusted using the Abbott formula (Abbott, 1925). Mortality data were assessed by probit analysis (Finney, 1971). According to the regression equation, the probability of mortality value corresponds to the y-axis, whereas the tested concentration corresponds to the x-axis. To obtain the LC50, we selected the concentrations that presented larval mortality above 50%. We used the Polo Plus software (Robertson et al., 2003) and a 95% confidence interval (CI); results with p<0.05 were considered significant.


The larvae of both species tested were sensitive to benzoyl thiosemicarbazone and to the metal nickel complex after 24 and 48 h of exposure. Table 1 shows that A. aegypti larvae were more susceptible to the benzoyl thiosemicarbazone than the nickel complex. The LC50 for the benzoyl thiosemicarbazone was 42.09 µg/mL, whereas for the nickel complex, it was 42.28 µg/mL. However, after 48 h, the metal complex showed the highest toxicity, with an LC50 of 35.84 µg/mL. In this case, the benzoyl thiosemicarbazone presented an LC50 of 38.49 g/L.
A. darlingi larvae were more susceptible than A. aegypti larvae for the compounds tested. LC50 values ​​for A. darlingi were lower at both the 24 and 48 h intervals (Table 2). Benzoyl thiosemicarbazone showed an LC50 of 4.77 µg/mL, whereas the nickel complex showed an LC50 of 7.33 µg/mL, both after 24 h. After 48 h, the LC50 of the metal complex was lower (2.68 µg/mL) than the LC50 of benzoyl thiosemicarbazone (2.72 µg/mL). The substance benzoyl thiosemicarbazone presented higher toxicity for both species tested after 24 h. Due to the sensitivity of the larvae, A. darlingi was more susceptible than A. aegypti larvae.


The present study addresses the larvicidal activity of a thiosemicarbazone derivative and its Ni complex against A.  aegypti and  A. darlingi,  species  responsible  for  the  transmission of dengue and malaria, respectively, in the Amazon region. Although the biological properties of the metal complexes of thiosemicarbazones have high toxicity associated with the free ligand (Mendes et al., 2006; Netalkar et al., 2015), we observed that after 24 h, the metal nickel complex was less toxic; that is, this complex displayed a higher lethal concentration against A. aegypti (LC50 42.28 µg/L) and A. darlingi (7.33 µg/L) than benzoyl thiosemicarbazone, which displayed an LC50 of 42.09 and 4.77 µg/mL, respectively.
After 48 h, however, the nickel complex showed better toxicity results, with an LC50 of 35.84 µg/mL for A. Aegypti and 2.68 µg/mL for A. darlingi. After the same period, benzoyl thiosemicarbazone presented an LC50 of 38.49 µg/mL for the first species and 2.72 µg/mL for the second.
Gopinathan and Arumugham (2015) evaluated the larvicidal activity of four Cu(II) metal complexes against Culex quinquefasciatus (LC50 0.61 to 2.09 mg/L) and Anopheles subpictus (LC50 0.89 to 1.88 mg/L). Although all complexes showed high toxicity, urea and thiourea complexed with copper presented the best larvicidal activity results when compared to thiosemicarbazide and semicarbazide. Thiosemicarbazide derivatives showed a broad spectrum of larvicidal activity at different concentrations.
Rayms-Keller et al. (1998) showed that metal ions were highly toxic to A. aegypti. For example, copper edetate in nanostructures and chitosan microcapsules showed efficacy against A. aegypti larvae, with an LC90 of 60 and 20 mg/L, respectively, because nanostructures and microcapsules favour the slow and continuous release of the active ingredient to the environment. Thus, when complexed to the nickel, thiosemicarbazone derivative tested here against A. aegypti and A. darling showed high toxicity. However, the results of this study indicate that the metal-ligand bond did not significantly favour larvicidal activity at all reading ranges, as observed for the assays against A. darlingi (Table 2); that is, the metal complex did not directly affect the simultaneous ion exchange in the biological system due to the reactivity and especially  the  redox  effect   caused   by   transition metals in biological systems (Stohs and Bagchi, 1995; Nguyen et al., 2000).
Beraldo and Gambino (2004) and Al-Amiery et al. (2012) described a series of thiosemicarbazone derivatives and metal complexes with different chemical and biological properties and highlighted the high biological potential of the metal complex relative to the free ligand. The compounds synthesized and evaluated against mosquito larvae in the present study need to be evaluated for toxicity against nontarget insects and especially regarding the accumulation of heavy metals in the environment, which requires special treatment for their removal (Mireji et al., 2010). The search for new active compounds is challenging because of the many cases of insects resistant to the insecticides currently used in vector control campaigns (Rose, 2001). Integrated mosquito management programmes targeting larvae and mosquitoes serve as one of the most effective ways to control insect populations and consequently reduce the number of vector-borne diseases in endemic areas.


Benzoyl thiosemicarbazone and its Ni(II) complex showed larvicidal activity against the larvae of A. aegypti and A. darlingi, indicating that the thiosemicarbazone metal complex has insecticidal potential. However, elucidating the mode of action of these compounds in larvae and developing new compounds with pharmacological potential are necessary.



The authors have not declared any conflict of interests.



The authors are grateful for the funding received from the National Institute of Amazonian Research (INPA);  the Federal Institute of Education, Science and Technology of Amazonas (IFAM); and the Malaria Network Project (Projeto Rede Malaria).


Abbott WS (1925). A method of computing the effectiveness of the insecticide. Journal of Economic Entomology 18:265-267.


Al-Amiery AA, Kadhum AAH, Mohamad AB (2012). Antifungal and antioxidant activities of pyrrolidone thiosemicarbazone complexes. Bioinorganic Chemistry and Applications 2012:1-6.


Arnold WR, Santore RC, Cotsifas JS (2005). Predicting copper toxicity in estuarine and marine waters using the Biotic Ligand Model. Marine Pollution Bulletin 50:1634-1640.


Beraldo H, Boyd LP, West DX (1997). Copper (II) and nickel (II) complexes of glyoxaldehyde bis {N (3)-substituted thiosemicarbazones}. Transition Metal Chemistry 23(1):67-71.


Beraldo H, Gambino D (2004). The wide pharmacological versatility of semicarbazones, thiosemicarba-zones and their metal complexes. Mini Reviews in Medicinal Chemistry 4:31-39.


Braga SF, Fonseca NC, Ramos JP, Souza-Fagundes EM, Oliveira RB (2016). Synthesis and cytotoxicity evaluation of thiosemicarbazones and their thiazole derivatives. Brazilian Journal of Pharmaceutical Sciences 52(2):299-308.


Chellan P, Nasser S, Vivas L, Chibale K, Smith GS (2010). Cyclopalladated complexes containing tridentate thiosemicarbazone ligands of biological significance: Synthesis, structure and antimalarial activity. Journal of Organometallic Chemistry 695(19):2225-2232.


Deane LM (1986). Malaria vectors in Brazil. Memórias do Instituto Oswaldo Cruz 81(2):5-14.


Despaigne AA, Vieira LF, Mendes IC, Costa FB, Speziali NL, Beraldo H (2010). Organotin(IV) complexes with 2-acetylpyridine benzoyl hydrazones: antimicrobial activity. Journal of the Brazilian Chemical Society 21(7):1247-1257.


Dulmage HT, Yousten AA, Singer SL, Lacey LA (1990). Guidelines for production of Bacillus thuringiensis H-14 and Bacillus sphaericus, Geneva. Available at: 



Finney DJ (1971). Probit analysis. Cambridge University Press Cambridge, England. Available at: 



Gopinathan H, Arumugham MN (2015). Larvicidal activity of synthesized copper(II) complexes against Culex quinquefasciatus and Anopheles subpictus. Journal of Taibah University for Science 9(1):27-33.


Greenbaum DC, Mackey Z, Hansell E, Doyle P, Gut J, Caffrey CR, Lehrman J, Rosenthal PJ, McKerrow JH, Chibale K (2004). Synthesis and Structure - Activity Relationships of Parasiticidal Thios emicarbazone Cysteine Protease Inhibitors against Plasmodium falciparum. Journal of Medicinal Chemistry 47(12):3212-3219.


Maciel-de-Freitas R, Aguiar R, Bruno RV, Guimarães MC, Lourenço-de-Oliveira R, Sorgine MH, Struchiner CJ, Valle D, O'Neill SL, Moreira LA (2012). Why do we need alternative tools to control mosquito-borne diseases in latin america? Memórias do Instituto Oswaldo Cruz 107(6):828-829.


Mendes IC, Moreira JP, Speziali NL, Mangrich AS, Takahashi JA, Beraldo H (2006). N(4)-tolyl-2-benzoylpyridine thiosemicarbazones and their copper(II) complexes with significant antifungal activity. Crystal structure of N(4)-para-tolyl-2- benzoylpyridine thiosemicarbazone. Journal of the Brazilian Chemical Society 17(8):1571-1577.


Mireji PO, Keating J, Hassanali A, Impoinvil DE, Mbogo CM, Muturi MN, Nyambaka H, Kenya EU, Githure JI, Beier JC (2010). Expression of metallothionein and α-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae). Ecotoxicology and Environmental Safety 73(1):46-50.


Nandal R, Deep A (2017). Metal Complexes with Antimalarial prospective: A review. Global Journal of Pharmaceutical Education and Research 6:22-27.


Nguyen TT, Ogwuru N, Eng G (2000). Tolerance of Aedes aegypti larvae to triorganotins. Applied Organometallic Chemistry 14(7):345-348.


Netalkar PP, Netalkar SP, Revankar VK (2015). Transition metal complexes of thiosemicarbazone: Synthesis, structures and in vitro antimicrobial studies. Polyhedron 100:215-222.


Parrilha GL, da Silva JG, Gouveia LF, Gasparoto AK, Dias RP, Rocha WR, Santos DA, Speziali NL, Beraldo H (2011). Pyridine-derived thiosemicarbazones and their tin(IV) complexes with antifungal activity against Candida spp. European Journal of Medicinal Chemistry 46(5):1473-1482


Pingaew R, Prachayasittikul S, Ruchirawat S (2010). Synthesis, cytotoxic and antimalarial activities of benzoyl thiosemicarbazone analogs of isoquinoline and related compounds. Molecules 15(2):988-996.


Puccioni-Sohler M, Roveroni N, Rosadas C, Ferry F, Peralta JM, Tanuri A (2017). Dengue infection in the nervous system: lessons learned for Zika and Chikungunya. Arquivos de Neuro-psiquiatria 75(2):123-126.


Rayms-Keller A, Olson KE, McGaw M, Oray C, Carlson JO, Beaty BJ (1998). Effect of heavy metals on Aedes aegypti (Diptera: Culicidae) larvae. Ecotoxicology and Environmental Safety 39(1):41-47.


Rebolledo AP, Vieites M, Gambino D, Piro OE, Castellano EE, Zani CL, Souza-Fagundes EM, Teixeira LR, Batista AA, Beraldo H (2005). Palladium(II) complexes of 2-benzoylpyridine-derived thiosemicarbazones: Spectral characterization, structural studies and cytotoxic activity. Journal of Inorganic Biochemistry 99(3):698-706.


Rivero A, Vezilier J, Weill M, Read AF, Gandon S (2010). Insecticide control of vector-borne diseases: When is insecticide resistance a problem? PLoS Pathogens 6(8):e1001000.


Robertson JL, Preisler HK, Russell RM (2007). PoloPlus: Probit and logit analysis user's guide. LeOra Software, Petaluna, CA, USA.


Rose RI (2001). Pesticides and public health: Integrated methods of mosquito management. Emerging Infectious Diseases 7(1):17-23.


Rosu T, Pahontu E, Pasculescu S, Georgescu R, Stanica N, Curaj A, Popescu A, Leabu M (2010). Synthesis, characterization antibacterial and antiproliferative activity of novel Cu(II) and Pd(II) complexes with 2-hydroxy-8-R-tricyclo[,7]tridecane-13-one thiosemicarbazone. European Journal of Medicinal Chemistry 45(4):1627-1634.


Simon F, Savini H, Parola P (2008). Chikungunya: A Paradigm of Emergence and Globalization of Vector-Borne Diseases. Medical Clinics of North America 92(6):1323-1343.


Silva JB, do AF Navarro DM, da Silva AG, Santos GK, Dutra KA, Moreira DR, Ramos MN (2015). Thiosemicarbazones as Aedes aegypti larvicidal. European Journal of Medicinal Chemistry 100:162-175.


Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, Mbogo CM, Hemingway J, Patil AP, Temperley WH, Gething PW (2012). A global map of dominant malaria vectors. Parasites and Vectors 5(1):69.


Stohs SJ, Bagchi D (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radical Biology and Medicine 18(2):321-336.


Tadei WP, Thatcher BD, Santos JM, Scarpassa VM, Rodrigues IB, Rafael MS (1998). Ecologic observations on anopheline vectors of malaria in the Brazilian amazon. The American Journal of Tropical Medicine and Hygiene 59(2):325-335.


Vi-uelas-Zahínos E, Luna-Giles F, Torres-García P, Fernández-Calderón MC (2011). Co(III), Ni(II), Zn(II) and Cd(II) complexes with 2-acetyl-2- thiazoline thiosemicarbazone: Synthesis, characterization, X-ray structures and antibacterial activity. European Journal of Medicinal Chemistry 46(1):150-159.


Wang Z, Ma Y, Xu Y, Ling Y, Yang X (2010). (E)-1-[(2-Chloro-5-methylpyridin-3-yl)methylene]thiosemicarbazide. Acta Crystallographica Section E: Structure Reports Online 66(3):o604-o604.


Wasi N, Singh HB (1987). Synthesis of metal complexes of antimalarial drugs and in-vitro evaluation of their activity against Plasmodium falciparum. Inorganica Chimica Acta 135(2):133-137.


World Health Organization (WHO) (2005). Guidelines for laboratory and field testing of mosquito larvicides, Geneva. Available at:


World Health Organization (WHO) (2014). Division of Malaria and Other Parasitic Diseases. From Malaria Control to Malaria Elimination: A Manual for Elimination Scenario Planning. World Health Organization, Geneva.


World Health Organization (WHO) (2017). World malaria report 2017. Geneva: World Health Organization; Licence: CC BY-NC-SA 3.0 IGO.


Xue CB, Zhang L, Luo WC, Xie XY, Jiang L, Xiao T (2007). 3D-QSAR and molecular docking studies of benzaldehyde thiosemicarbazone, benzaldehyde, benzoic acid, and their derivatives as phenoloxidase inhibitors. Bioorganic and Medicinal Chemistry 15(5):2006-2015.