**Antimycobacterial Activity Some Different Lamiaceae Plant Extracts Containing Flavonoids and Other Phenolic Compounds**

Tulin Askun, Gulendam Tumen, Fatih Satil, Seyma Modanlioglu and Onur Yalcin *Balikesir University Turkey* 

## **1. Introduction**

308 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Von Groll, A. Martin, A., Jureen, P., Hoffner, S., Vandamme, P. et al. (2009).

WHO (2001). World Health Organisation. Guidelines for drug susceptibility testing for

WHO (2011). *World Health Organisation. Progress report 2011.* (2011). Towards universal

and gyrB. *Antimicrob. Agents Chemother*., 53(10), pp. 4498-4500.

second-line anti-tuberculosis drugs for dots-plus.

resistant tuberculosis by 2015*.*

Fluoroquinolones resistance in *Mycobacterium tuberculosis* and mutations in gyrA

access to diagnosis and treatment of multidrug-resistant and extensively drug-

*Mycobacterium tuberculosis* is a pathogenic bacteria species of the genus *Mycobacterium*, first discovered in 1882 by Robert Koch, which causes tuberculosis (TB) (Ryan & Ray, 2004). The disease is characterized by symptoms such as sepsis, septic shock, multiple organ failure (Muckart & Bhagwanjee, 1997). It may spread to the central nervous system and cause TB meningitis, intracranial tuberculomas, or abscesses (Harisinghani et al., 2000; Hwang et al., 2010).

After the late 1980s, tuberculosis morbidity and mortality rates became a major health problem for industrialized countries (Raviglione et al., 1995; Heym & Cole, 1997). Multidrug-resistant tuberculosis (MDR TB) and extensively drug resistant tuberculosis (XDR TB) has become a common phenomenon, which cause drugs to be ineffective. MDR-TB results from either primary infection or may develop in the course of a patient's treatment. MDR TB is resistant to at least two first-line anti-TB drugs, isoniazid (INH) and rifampicin (RIF), which are most powerful anti-TB drugs; XDR TB is resistant to INH and RIF, plus fluoroquinolone and at least one of three injectable second-line drugs such as capreomycin, kanamycin, and amikacin. Treatment of XDR-TB is not possible by first-line anti-TB drugs, which are less effective, expensive and toxic; in addition treatment takes two years or more (WHO, 2011a; WHO, 2011b).

Mycobacteria are resistant to most common antibiotics and chemotherapeutic agents due to the mycobacterial cell wall composition of bacterial peptidoglycans (Slayden & Barry, 2000; Lee et al., 1996; Brennan et al., 1995), a lipophilic layer of long-chain fatty acids, and mycolic acids (Barry et al., 1998). The rich lipids of the cell wall has an important role in their virulence (Murray, Rosenthal and Pfaller, 2005). This structure provides a highly hydrophobic and efficient barrier to antibiotics and chemotherapeutic agents (Jarlier & Nikaido 1994). Thus, this cell wall composition restricts the choice of drug treatment. Compounds capable of blocking efflux pumps so that antibiotics can gain access to their targets are of obvious importance (Viveiros et al, 2003). Increased activity of existing efflux

Antimycobacterial Activity Some Different Lamiaceae

children who were in contact with adults.

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 311

2008b). Today, one of the most important global health problems is changes in behavior of TB, such as resistance to anti-TB drugs and the influence of the HIV epidemic (World Health Organization, 2008a). WHO Global TB Control (2009) reported that there were approximately 0.5 million cases of MDR-TB in 2007. The World Health Organization (2010) reported that there were 9.4 million new TB cases globally and approximately 1.7 million people died from TB. The organization also reported that 1.2 million people were living with HIV and 76% of these people were residing in the African region while 14% were living in the South East Asian region in 2009 (World Health Organization, 2010). In South Africa, TB is the most commonly notified disease and the fifth largest cause of death among the black population. The prevalence of TB continues to increase all over the world. Although the main reasons are known to be the human immunodeficiency virus (HIV) and the emergence of drug-resistant strains of TB (WHO, 2009), the other factors include poverty, drug addiction, inadequate health conditions and migration (Antunes et al., 2000; Merza et al., 2011). WHO reports (2011a) estimated that the risk of developing tuberculosis (TB) is between 20 and 37 times greater in people living with HIV than among the general population. In addition, infection with Human immunodeficiency virus type 1 (HIV-1) disrupts immunological control of *Mycobacterium* infections due to the loss of CD4+ T cells. Salte et al. (2011) reported that *Mycobacterium avium* is one of the most common opportunistic infections among AIDS patients. Snider et al. (1985) examined the transmission of MDR-TB strains from adult to child contacts and confirmed the progression of the disease by DNA fingerprint studies. INH-resistant strains caused much infection in

Mycobacteria are Gram-resistant non-motile pleomorphic rods with a waxy cell wall. These bacteria include high lipid content within the cell wall (Wilbur et al., 2009; Jackson et al., 2007), the complex lipids esterified with long-chain fatty acids. Myobacteria are referred to as acid fast Gram-positive due to their resistance to dilute acid and ethanol-based decolorization procedures and their lack of an outer cell membrane. When they are stained using concentrated dyes, combined with heat, they do not give up the color by the dilute

Some medicinal plants have been used to treat the symptoms of TB including *Acacia nilotica*, *Cassine papillosa*, C*henopodium ambrosioides*, *Combretum molle*, and *Euclea natalensis* from Africa (Watt and Breyer-Brandwijk, 1962; Pujol, 1990; Lall & Meyer, 2001; Bryant, 1966).

Natural products are an important source of new chemical compounds and, hopefully, therapeutic agents for many bacterial diseases. Lall and Meyer (1999) reported antimycobacterial activity of *Euclea natalensis* (Ebenaceae), which is rich in naphthoquinones, against drug-sensitive and drug-resistant strains of *M. tuberculosis.* Gordien et al. (2009) studied two terpenes, sesquiterpene and longifolene; and two diterpenes, totarol and transcommunic acid, obtained from the aerial parts and roots of *Juniperus communis*. They reported that totarol showed the highest activity against *Mycobacterium tuberculosis* H37Rv and that longifolene and totarol exhibited the most activity against rifampicin-resistant variants. Phenolic compounds have some effects on microbial metabolism and growth, depending on

Many studies have shown that phenolic compounds inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria (Davidson et al., 2005; Estevinho et al., 2008)

their concentration and active compounds (Alberto et al., 2001; Reguant et al., 2000).

acid and ethanol-based de-colorization procedures (Ryan & Ray, 2004).

pumps were caused by ineffective therapy of TB patients, which is develops bacterial resistancy to one or more drug. Recent researches showed that mycobacteria have multiple putative efflux pumps which is a key factor for gaining resistance (Braibant, 2000; De Rossi et al., 2002). In addition to, chromosomal gene mutation and then accumulation of these mutations also one of the origine of multidrug-resistant (Ramaswamy & Musser, 1998; Gillespie, 2002; Viveiros et al., 2003).

Some well-known drugs and their mechanism of actions affect bacteria in different ways. Streptomycin (STR) has been used to treat tuberculosis patients since the 1940s; INH was used to treat tuberculosis in the 1960s; RIF was first used at the beginning of the 1970s (Toungoussova et al., 2006); and ethambutol (EMB) was introduced in 1961 as a bacteriostatic first-line drug (Perdigão et al., 2009). RIF inhibits transcription to RNA and translation to proteins by binding its' beta subunit of RNA polymerase in bacteria; however, if bacteria produce a different beta subunit, they are not affected by the drug (O'Sullivan et al., 2005). STR is a protein synthesis inhibitor. STR interacts with a 30S subunit of ribosome and disrupts protein synthesis (Sharma et al., 2007; Springer et al., 2001). Its mechanism of action starts with binding tightly to the phosphate backbone of 16S rRNA in different domains and making contact with the S12 ribosomal protein; finally it causes misreading of the bacterial genetic code during translation (Carter et al., 2000; Hosaka et al., 2006). INH is activated by an enzyme, catalase-peroxidase, called KatG in *M. tuberculosis*. KatG, isonicotinic acyl and NADH form a complex that binds enoyl-acyl carrier protein reductase (InhA) and affects fatty acid synthase. The identification of an enoyl-acyl carrier protein (ACP) reductase plays a role in INH resistance named InhA. In this way, mycolic acid synthesis and cell wall development are inhibited (van Veen & Konings, 1998; Slayden & Barry, 2000; Suarez et al., 2009). As a result, when exposed to INH, Mycobacteria lose their acid-fastness and viability. Changes in the catalaseperoxidase gene (katG) and the inhA genes have been defined as one of the mechanisms of drug resistance in *M. tuberculosis* (Morris et al., 1995; Heym et al., 1995; Mohamad et al., 2004). EMB is a potent synthetic antimycobacterial agent that may cause optic neuropathy in patients (Kozak et al., 1998).

EMB has a bacteriostatic effect and interferes with mycolic acid synthesis, phospholipid metabolism, and arabinogalactan synthesis (Kilburn et al., 1977; Takayama & Kilburn, 1989) and affects nucleic acid metabolism (Forbes et al.,, 1965). EMB has synergistic actions, when combined with other agents, against *Mycobacterium avium* (Inderlied and Salfinger, 1995). TB is currently one of the most serious infectious diseases all over the world. Antimycobacterial drugs cause unpleasant side effects and trigger changes in the antibiotic target, thereby reducing the efficacy of drug therapies. Mycobacteria have recently increased their virulence and tuberculosis (TB) is the most lethal infection in the world. Between 1980 and 2005, 90 million cases of TB worldwide were reported to the WHO (World Health Organization) and over three in every thousand people die of TB, which is the highest rate in the world (Lall and Meyer, 1999). Yang et al. (2010) also reported that the prevalence of MDR-TB among the Chinese people has increased since 1985. The WHO stated, ''The global incidence of TB was estimated to be 136 cases per 100,000 population per year in 2005. In addition, the WHO region of the Americas and the WHO African region represent a total of 8.8 million new cases of TB and 1.6 million deaths from TB every year" (World Health Organization, 2008a). There were 9.5 million TB-related child deaths globally in 2006 (World Health Organization,

pumps were caused by ineffective therapy of TB patients, which is develops bacterial resistancy to one or more drug. Recent researches showed that mycobacteria have multiple putative efflux pumps which is a key factor for gaining resistance (Braibant, 2000; De Rossi et al., 2002). In addition to, chromosomal gene mutation and then accumulation of these mutations also one of the origine of multidrug-resistant (Ramaswamy & Musser, 1998;

Some well-known drugs and their mechanism of actions affect bacteria in different ways. Streptomycin (STR) has been used to treat tuberculosis patients since the 1940s; INH was used to treat tuberculosis in the 1960s; RIF was first used at the beginning of the 1970s (Toungoussova et al., 2006); and ethambutol (EMB) was introduced in 1961 as a bacteriostatic first-line drug (Perdigão et al., 2009). RIF inhibits transcription to RNA and translation to proteins by binding its' beta subunit of RNA polymerase in bacteria; however, if bacteria produce a different beta subunit, they are not affected by the drug (O'Sullivan et al., 2005). STR is a protein synthesis inhibitor. STR interacts with a 30S subunit of ribosome and disrupts protein synthesis (Sharma et al., 2007; Springer et al., 2001). Its mechanism of action starts with binding tightly to the phosphate backbone of 16S rRNA in different domains and making contact with the S12 ribosomal protein; finally it causes misreading of the bacterial genetic code during translation (Carter et al., 2000; Hosaka et al., 2006). INH is activated by an enzyme, catalase-peroxidase, called KatG in *M. tuberculosis*. KatG, isonicotinic acyl and NADH form a complex that binds enoyl-acyl carrier protein reductase (InhA) and affects fatty acid synthase. The identification of an enoyl-acyl carrier protein (ACP) reductase plays a role in INH resistance named InhA. In this way, mycolic acid synthesis and cell wall development are inhibited (van Veen & Konings, 1998; Slayden & Barry, 2000; Suarez et al., 2009). As a result, when exposed to INH, Mycobacteria lose their acid-fastness and viability. Changes in the catalaseperoxidase gene (katG) and the inhA genes have been defined as one of the mechanisms of drug resistance in *M. tuberculosis* (Morris et al., 1995; Heym et al., 1995; Mohamad et al., 2004). EMB is a potent synthetic antimycobacterial agent that may cause optic neuropathy

EMB has a bacteriostatic effect and interferes with mycolic acid synthesis, phospholipid metabolism, and arabinogalactan synthesis (Kilburn et al., 1977; Takayama & Kilburn, 1989) and affects nucleic acid metabolism (Forbes et al.,, 1965). EMB has synergistic actions, when combined with other agents, against *Mycobacterium avium* (Inderlied and Salfinger, 1995). TB is currently one of the most serious infectious diseases all over the world. Antimycobacterial drugs cause unpleasant side effects and trigger changes in the antibiotic target, thereby reducing the efficacy of drug therapies. Mycobacteria have recently increased their virulence and tuberculosis (TB) is the most lethal infection in the world. Between 1980 and 2005, 90 million cases of TB worldwide were reported to the WHO (World Health Organization) and over three in every thousand people die of TB, which is the highest rate in the world (Lall and Meyer, 1999). Yang et al. (2010) also reported that the prevalence of MDR-TB among the Chinese people has increased since 1985. The WHO stated, ''The global incidence of TB was estimated to be 136 cases per 100,000 population per year in 2005. In addition, the WHO region of the Americas and the WHO African region represent a total of 8.8 million new cases of TB and 1.6 million deaths from TB every year" (World Health Organization, 2008a). There were 9.5 million TB-related child deaths globally in 2006 (World Health Organization,

Gillespie, 2002; Viveiros et al., 2003).

in patients (Kozak et al., 1998).

2008b). Today, one of the most important global health problems is changes in behavior of TB, such as resistance to anti-TB drugs and the influence of the HIV epidemic (World Health Organization, 2008a). WHO Global TB Control (2009) reported that there were approximately 0.5 million cases of MDR-TB in 2007. The World Health Organization (2010) reported that there were 9.4 million new TB cases globally and approximately 1.7 million people died from TB. The organization also reported that 1.2 million people were living with HIV and 76% of these people were residing in the African region while 14% were living in the South East Asian region in 2009 (World Health Organization, 2010). In South Africa, TB is the most commonly notified disease and the fifth largest cause of death among the black population. The prevalence of TB continues to increase all over the world. Although the main reasons are known to be the human immunodeficiency virus (HIV) and the emergence of drug-resistant strains of TB (WHO, 2009), the other factors include poverty, drug addiction, inadequate health conditions and migration (Antunes et al., 2000; Merza et al., 2011). WHO reports (2011a) estimated that the risk of developing tuberculosis (TB) is between 20 and 37 times greater in people living with HIV than among the general population. In addition, infection with Human immunodeficiency virus type 1 (HIV-1) disrupts immunological control of *Mycobacterium* infections due to the loss of CD4+ T cells. Salte et al. (2011) reported that *Mycobacterium avium* is one of the most common opportunistic infections among AIDS patients. Snider et al. (1985) examined the transmission of MDR-TB strains from adult to child contacts and confirmed the progression of the disease by DNA fingerprint studies. INH-resistant strains caused much infection in children who were in contact with adults.

Mycobacteria are Gram-resistant non-motile pleomorphic rods with a waxy cell wall. These bacteria include high lipid content within the cell wall (Wilbur et al., 2009; Jackson et al., 2007), the complex lipids esterified with long-chain fatty acids. Myobacteria are referred to as acid fast Gram-positive due to their resistance to dilute acid and ethanol-based decolorization procedures and their lack of an outer cell membrane. When they are stained using concentrated dyes, combined with heat, they do not give up the color by the dilute acid and ethanol-based de-colorization procedures (Ryan & Ray, 2004).

Some medicinal plants have been used to treat the symptoms of TB including *Acacia nilotica*, *Cassine papillosa*, C*henopodium ambrosioides*, *Combretum molle*, and *Euclea natalensis* from Africa (Watt and Breyer-Brandwijk, 1962; Pujol, 1990; Lall & Meyer, 2001; Bryant, 1966).

Natural products are an important source of new chemical compounds and, hopefully, therapeutic agents for many bacterial diseases. Lall and Meyer (1999) reported antimycobacterial activity of *Euclea natalensis* (Ebenaceae), which is rich in naphthoquinones, against drug-sensitive and drug-resistant strains of *M. tuberculosis.* Gordien et al. (2009) studied two terpenes, sesquiterpene and longifolene; and two diterpenes, totarol and transcommunic acid, obtained from the aerial parts and roots of *Juniperus communis*. They reported that totarol showed the highest activity against *Mycobacterium tuberculosis* H37Rv and that longifolene and totarol exhibited the most activity against rifampicin-resistant variants. Phenolic compounds have some effects on microbial metabolism and growth, depending on their concentration and active compounds (Alberto et al., 2001; Reguant et al., 2000).

Many studies have shown that phenolic compounds inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria (Davidson et al., 2005; Estevinho et al., 2008)

Antimycobacterial Activity Some Different Lamiaceae

through a syringe filter (Sartorius, Ø 0.22 µm.) before use.

**2.2 Preparation of plant extracts** 

**2.3 Chemicals and samples** 

injection into the capillary.

**2.5 Preparation of standards** 

**2.4 LC-MS conditions** 

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 313

The plants [*O. acutidens* (60 g), *O. sipyleum* (66 g), *Salvia viridis* (12 g), *S. microstegia* (100 g), *Satureja boissieri* (101 g), *Stachys byzantina* (65 g)*, S. cretica* (37 g)*, S. cretica* subsp *smyrnaea* (71 g), *T. syriacus* (44 g)*,* and *T. cilicicus (*85 g) (endemic)], were air-dried at room temperature. Extracts of dried plants were prepared by the sequential extraction method (Chan et al., 2008) using 1 L of chloroform (CL), ethyl acetate (EA) and methanol (ME) at room temperature over a period of fifteen days. Finally, three extract fractions were obtained from each plants. The extracts were filtered through filter paper concentrated using a rotary evaporator and dried in vacuo at 40 ºC. They were stored at −20◦C until use. The total yields from chloroform (CL), ethyl acetate (EA) and methanol (ME) extracts were *O. acutidens* (0.57, 0.74, 4.88g), *O. sipyleum (*2.14, 1.61, 5.50g), *Salvia viridis* (0.22, 0.15, 1.56 g), *S. microstegia* (8.17, 0.77, 6.70g), *Satureja boissieri* (4.05, 1.08, 8.82g), *Stachys byzantina (*7.69, 1.10, 5.15g), *S. cretica (*1.16, 0.59, 3.47g), *S. cretica* subsp*. smyrnaea (*1.92, 1.43, 6.92g), *T. syriacus (*1.80, 1.08, 2.85g), and *T. cilicicus (*2.37, 3.18, 5.35g) respectively. All stocks were stored at -20 ºC. To conduct antimicrobial activity tests, samples were dissolved in dimethyl sulfoxide (DMSO) and prepared at a concentration of 100 mg/mL. All the extracts used were sterilized by passing

Gradient grade MeOH and acetonitrile were purchased from MERCK. Gradient grade water (18m) was prepared using a Purelab Option-Q elga dv25 system. All standard stock solutions (1 mg/mL) were prepared by dissolving each compound in MeOH. Standards, rosmarinic acid, trans cinnamic acid, and ferulic acid were purchased from Aldrich, caffeic acid and gallic acid from Sigma-Aldrich and all other chemicals used were obtained from Sigma. All solutions were filtered through a membrane filters (Sartorius, Ø 0.22 µm.) before

Analyses were performed with Agilent LC-MS system (1200 LC with a single quadrupole) with ESI source negative mode. Source parameters were optimized to provide highest sensitivity. The source parameters are: Gas temperature 350 °C, drying gas flow 12 l/min, nebulizer pressure 50 psi, capillary voltage 3500 V., seperation was carried by a C-18 column (EC-C18 4,6x50mm 2.7um). Mobile phases are A: Water (5 mM ammonium formate+ 0.5 % formic acid) and B (acetonitrile). The gradient program is: 5 % B for starting condition and increased up to 45 % B in 1 min, hold 2 min, increase % B to 95 from 3 to 6 min, hold 1 min and decrease % B to 5% at final step. Total run time is 12 min. Injection volume is 5 µl. The detection was accomplished using MS SIM mode. Scan mode is also used. The LC–MS

Twenty standards were used for quantitative and qualitative determination: trans-cinnamic acid [(Rt) 4.98 min], ρ-coumeric acid (Rt 3.95 min), vanillic acid (Rt 3.79 min), gallic acid (Rt 1.89 min), caffeic acid (Rt 3.72 min), ferulic acid (Rt 3.99 min), ), apigenin (Rt 4.83 min), naringenin (Rt 4.85 min), luteolin (Rt 4.43 min), epicatechin (Rt 3.67 min), quercetin (Rt 4.42

analysis was based in a method described by Pérez-Magariño et al. (1999).

Flavonoids are the most common group of polyphenolic compounds. Flavonoids are plant secondary metabolites with a fused ring system, which are found as glycosides in plants. Of the well-known flavonoids, apigenin has a calming effect, while quercetin and kaempferol have a sedative effect (Jäger & Saaby, 2011).

In previous studies, flavonoids were reported to show antimicrobial (Cushnie & Lamb, 2006, 2011), anti-allergic (Chen et al., 2010), anti-inflammatory (Seo et al., 2000), and anticarcinogenic (Lee et al., 2008) activities. Until 2004, it was suggested (Cushnie and Lamb, 2005, 2011) that their antibacterial efficacy was dependent upon cytoplasmic membrane damage by perforation (Ikigai et al., 1993), inhibition of nucleic acid synthesis (Mori et al., 1987) and disruption of energy metabolism due to NADH-cytochrome c reductase inhibition (Haraguchi et al., 1998). Currently, some other supporting mechanisms have emerged to indicate the role of flavonoids in antibacterial activity; these mechanisms include damage to the cytoplasmic membrane by generating hydrogen peroxide (Tamba et al., 2007; Kusuda et al., 2006; Sirk et al., 2008), inhibition of nucleic acid synthesis (Gradisar et al., 2007; Wang et al., 2010) and inhibition of ATP synthase (Chinnam et al., 2010). While Puupponen-Pimiä et al. (2001) reported that catechin, rutin and quercetin did not affect the growth of *E. coli*, Vaquero et al., (2007) reported that quercetin was the strongest inhibitor active against bacteria, dependent on concentration.

Lamiaceae, also known as mint, is a family of flowering plants that includes 250 to 258 genera and approximately 6,000 to 6,970 species across the world (Zomlefer, 1994; Mabberley, 1997). The family has a cosmopolitan distribution and contains many plant species with culinary and medicinal purposes; examples of the former are basil, mint, rosemary, sage, savory, marjoram, oregano, thyme, lavender, and perilla (Naghibi et al., 2005). The Lamiaceae family of plants have been used since ancient times as folk remedies for various health problems such as common cold, throat infections, acaricidal, psoriasis, seborrheic eczema, hemorrhage, menstrual disorders, miscarriage, ulcer, spasm and stomach problems (Takayama et al., 2011; Loizzo et al., 2010;. Ribeiro et al., 2010). Their constituents, particularly diterpenoids and triterpenoids, have been found to have antiseptic, antibacterial, anti-inflammatory, cytotoxic, cardio-active and other properties (Ulubelen, 2003).

In our previous studies, we tested more than 100 plant extracts, some of which showed antimycobacterial activity against *Mycobacterium tuberculosis*. In this study, in the light of our past experiences, we present a continuation of the testing of some of the plant extracts and the efficacy of their antimycobacterial properties.

## **2. Materials & methods**

#### **2.1 Plant materials**

Aerial parts (herbs in the flowering stage) of plants, *Origanum acutidens* (Hand.-Mazz.) Ietswaart, *Origanum sipyleum* L., *Salvia viridis* L., *Salvia microstegia* Boiss&Bal., *Satureja boissieri* Hausskn. ex Boiss., *Stachys byzantina* C.Koch., *Stachys cretica* L., *Stachys cretica* subsp. *smyrnaea*  Rech. fil., *Thymus syriacus* Boiss., and *Thymus cilicicus* Boiss&Bal.*(endemic)* were collected from different parts of Turkey between 2009 and 2010. The plants were identified by Assoc. Prof. Dr. F. Satil at Balkesir University, Turkey. Voucher specimens were deposited in the herbarium of Balikesir University Department of Biology. Herbarium plant data, such as locality, altitude, and collection time and identification number of species are given in Table 1.

#### **2.2 Preparation of plant extracts**

312 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Flavonoids are the most common group of polyphenolic compounds. Flavonoids are plant secondary metabolites with a fused ring system, which are found as glycosides in plants. Of the well-known flavonoids, apigenin has a calming effect, while quercetin and kaempferol

In previous studies, flavonoids were reported to show antimicrobial (Cushnie & Lamb, 2006, 2011), anti-allergic (Chen et al., 2010), anti-inflammatory (Seo et al., 2000), and anticarcinogenic (Lee et al., 2008) activities. Until 2004, it was suggested (Cushnie and Lamb, 2005, 2011) that their antibacterial efficacy was dependent upon cytoplasmic membrane damage by perforation (Ikigai et al., 1993), inhibition of nucleic acid synthesis (Mori et al., 1987) and disruption of energy metabolism due to NADH-cytochrome c reductase inhibition (Haraguchi et al., 1998). Currently, some other supporting mechanisms have emerged to indicate the role of flavonoids in antibacterial activity; these mechanisms include damage to the cytoplasmic membrane by generating hydrogen peroxide (Tamba et al., 2007; Kusuda et al., 2006; Sirk et al., 2008), inhibition of nucleic acid synthesis (Gradisar et al., 2007; Wang et al., 2010) and inhibition of ATP synthase (Chinnam et al., 2010). While Puupponen-Pimiä et al. (2001) reported that catechin, rutin and quercetin did not affect the growth of *E. coli*, Vaquero et al., (2007) reported that quercetin was the strongest inhibitor active against

Lamiaceae, also known as mint, is a family of flowering plants that includes 250 to 258 genera and approximately 6,000 to 6,970 species across the world (Zomlefer, 1994; Mabberley, 1997). The family has a cosmopolitan distribution and contains many plant species with culinary and medicinal purposes; examples of the former are basil, mint, rosemary, sage, savory, marjoram, oregano, thyme, lavender, and perilla (Naghibi et al., 2005). The Lamiaceae family of plants have been used since ancient times as folk remedies for various health problems such as common cold, throat infections, acaricidal, psoriasis, seborrheic eczema, hemorrhage, menstrual disorders, miscarriage, ulcer, spasm and stomach problems (Takayama et al., 2011; Loizzo et al., 2010;. Ribeiro et al., 2010). Their constituents, particularly diterpenoids and triterpenoids, have been found to have antiseptic, antibacterial, anti-inflammatory, cytotoxic,

In our previous studies, we tested more than 100 plant extracts, some of which showed antimycobacterial activity against *Mycobacterium tuberculosis*. In this study, in the light of our past experiences, we present a continuation of the testing of some of the plant extracts and

Aerial parts (herbs in the flowering stage) of plants, *Origanum acutidens* (Hand.-Mazz.) Ietswaart, *Origanum sipyleum* L., *Salvia viridis* L., *Salvia microstegia* Boiss&Bal., *Satureja boissieri* Hausskn. ex Boiss., *Stachys byzantina* C.Koch., *Stachys cretica* L., *Stachys cretica* subsp. *smyrnaea*  Rech. fil., *Thymus syriacus* Boiss., and *Thymus cilicicus* Boiss&Bal.*(endemic)* were collected from different parts of Turkey between 2009 and 2010. The plants were identified by Assoc. Prof. Dr. F. Satil at Balkesir University, Turkey. Voucher specimens were deposited in the herbarium of Balikesir University Department of Biology. Herbarium plant data, such as locality, altitude,

and collection time and identification number of species are given in Table 1.

have a sedative effect (Jäger & Saaby, 2011).

bacteria, dependent on concentration.

cardio-active and other properties (Ulubelen, 2003).

the efficacy of their antimycobacterial properties.

**2. Materials & methods** 

**2.1 Plant materials** 

The plants [*O. acutidens* (60 g), *O. sipyleum* (66 g), *Salvia viridis* (12 g), *S. microstegia* (100 g), *Satureja boissieri* (101 g), *Stachys byzantina* (65 g)*, S. cretica* (37 g)*, S. cretica* subsp *smyrnaea* (71 g), *T. syriacus* (44 g)*,* and *T. cilicicus (*85 g) (endemic)], were air-dried at room temperature. Extracts of dried plants were prepared by the sequential extraction method (Chan et al., 2008) using 1 L of chloroform (CL), ethyl acetate (EA) and methanol (ME) at room temperature over a period of fifteen days. Finally, three extract fractions were obtained from each plants. The extracts were filtered through filter paper concentrated using a rotary evaporator and dried in vacuo at 40 ºC. They were stored at −20◦C until use. The total yields from chloroform (CL), ethyl acetate (EA) and methanol (ME) extracts were *O. acutidens* (0.57, 0.74, 4.88g), *O. sipyleum (*2.14, 1.61, 5.50g), *Salvia viridis* (0.22, 0.15, 1.56 g), *S. microstegia* (8.17, 0.77, 6.70g), *Satureja boissieri* (4.05, 1.08, 8.82g), *Stachys byzantina (*7.69, 1.10, 5.15g), *S. cretica (*1.16, 0.59, 3.47g), *S. cretica* subsp*. smyrnaea (*1.92, 1.43, 6.92g), *T. syriacus (*1.80, 1.08, 2.85g), and *T. cilicicus (*2.37, 3.18, 5.35g) respectively. All stocks were stored at -20 ºC. To conduct antimicrobial activity tests, samples were dissolved in dimethyl sulfoxide (DMSO) and prepared at a concentration of 100 mg/mL. All the extracts used were sterilized by passing through a syringe filter (Sartorius, Ø 0.22 µm.) before use.

#### **2.3 Chemicals and samples**

Gradient grade MeOH and acetonitrile were purchased from MERCK. Gradient grade water (18m) was prepared using a Purelab Option-Q elga dv25 system. All standard stock solutions (1 mg/mL) were prepared by dissolving each compound in MeOH. Standards, rosmarinic acid, trans cinnamic acid, and ferulic acid were purchased from Aldrich, caffeic acid and gallic acid from Sigma-Aldrich and all other chemicals used were obtained from Sigma. All solutions were filtered through a membrane filters (Sartorius, Ø 0.22 µm.) before injection into the capillary.

#### **2.4 LC-MS conditions**

Analyses were performed with Agilent LC-MS system (1200 LC with a single quadrupole) with ESI source negative mode. Source parameters were optimized to provide highest sensitivity. The source parameters are: Gas temperature 350 °C, drying gas flow 12 l/min, nebulizer pressure 50 psi, capillary voltage 3500 V., seperation was carried by a C-18 column (EC-C18 4,6x50mm 2.7um). Mobile phases are A: Water (5 mM ammonium formate+ 0.5 % formic acid) and B (acetonitrile). The gradient program is: 5 % B for starting condition and increased up to 45 % B in 1 min, hold 2 min, increase % B to 95 from 3 to 6 min, hold 1 min and decrease % B to 5% at final step. Total run time is 12 min. Injection volume is 5 µl. The detection was accomplished using MS SIM mode. Scan mode is also used. The LC–MS analysis was based in a method described by Pérez-Magariño et al. (1999).

#### **2.5 Preparation of standards**

Twenty standards were used for quantitative and qualitative determination: trans-cinnamic acid [(Rt) 4.98 min], ρ-coumeric acid (Rt 3.95 min), vanillic acid (Rt 3.79 min), gallic acid (Rt 1.89 min), caffeic acid (Rt 3.72 min), ferulic acid (Rt 3.99 min), ), apigenin (Rt 4.83 min), naringenin (Rt 4.85 min), luteolin (Rt 4.43 min), epicatechin (Rt 3.67 min), quercetin (Rt 4.42

Antimycobacterial Activity Some Different Lamiaceae

used for the inoculation procedures.

**2.8 Antimycobacterial activity test** 

cultures reincubated in fresh medium.

**2.8.2 Determination of mycobactericidal activity** 

chromatograms of standards (Figs. 1–3).

concentration (MBC).

reincubated in fresh medium.

*tuberculosis* 

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 315

To prepare *M. tuberculosis* inoculum using a BACTEC MGIT tube with positive growth, the positive tubes were used beginning from the day after the sample first became positive (day-1 positive), up to and including the fifth day (day-5 positive). The positive tubes that were older than five days were subcultured into fresh growth medium. Tubes that were day-1 and day-2 positive were used in the inoculation procedure for the susceptibility tests. The tubes that were between day-3 and day-5 positive were diluted using 1 mL of the positive broth and 4 mL of sterile saline solution; the 5 mL diluted suspension samples were

Antimycobacterial bioassay was performed using the Microplate Alamar Blue Assay (MABA) method (Collins and Franzblau, 1997). MIC was recorded as the lowest drug concentration that prevented to turn blue to pink colour by adding Alamar blue. MBC was also recorded the minimum extract concentration that do not cause any color changing in

**2.8.1 Determination of Minimal Inhibitory Concentrations (MICs) for** *Mycobacterium* 

Microplates were inoculated with the bacterial suspension (20 μL per well except for the negative control wells) and incubated at 37 °C for 6 days. Alamar blue (15 μL, Trek Diagnostic system) was then added to the bacterial growth control wells (without extract) and the microplates were incubated at 37 °C for an additional 24 hours. If the dye turned from blue to pink, (indicating positive bacterial growth) then Alamar blue solution was added to the other wells to determine the MIC values. All tests were performed in triplicate.

All the extracts prepared from aerial parts of plants, the herbarium data of these species shown in Table 1, were analyzed by LC-MS. The quantity of chemicals in the methanol extracts are given in Table 2. Chromatograms of phenols in all extracts were compared to

The plant extracts described above were used in mycobactericidal activity tests. Two-fold dilution series in triplicate sets of parallel microplate wells were used for each extract. To determine the minimum bactericide concentrations (MBCs), fresh Middlebrook 7H9 culture broth (185 μL) was transferred to each well. A fifteen microliter of an Mycobacterial suspension, from MIC concentration and higher concentration wells obtained from the MIC test described above was added to each well, in order to determine the minimum bactericide

Two microplate wells were used as positive and negative controls, and each test was repeated in triplicate. For the negative controls, 200 mL of fresh broth (Middlebrook 7H9 culture medium and OADC) was used. For positive controls, including 185 μL and inoculums from former positive control wells (15 μL) was used. After 24 hours of incubation and colour development using the Alamar blue solution, MBCs were recorded as the minimum extract concentration that did not cause any colour change in cultures when

min), carnosic acid (Rt. 8.55 min), chlorogenic acid (Rt 3.59 min), rosmarinic acid (Rt 3.97 min), apigenin 7-glucoside (Rt 3.89 min), oleuropein (Rt 3.969 min), amentoflavone (Rt 5.16 min), naringin (Rt 3.83 min), rutin hydrate (Rt 3.69 min), hesperidin (Rt 3.85 min). Calibration concentrations were 1,4,5 and 20 ppm except one, apigenin 7-glucoside, was 0.9, 1.8, 4.5, 9, and 18 ppm and injection volume was 5 µL for all standards.

## **2.6 Organisms**

The extracts were screened against four strain, *M. tuberculosis* H37Ra (ATCC 25177), *M. tuberculosis* H37Rv (ATCC 25618) and two-positive *M. tuberculosis* isolates obtained from patient from hospital, for antibacterial activity.

## **2.7 Preparation of** *Mycobacterium tuberculosis* **inocula**

Bacterial suspensions of *M. tuberculosis* were prepared either from Lowenstein–Jensen slants or from complete 7H9 broth cultures. To prepare an inoculum that was less than 15 days old from a culture grown on Lowenstein-Jensen medium, a suspension was prepared in Middlebrook 7H9 broth. The turbidity of the suspension was adjusted to a 1.0 McFarland standard. The suspension was vortexed for several minutes and was allowed to stand for 20 min for the initial settling of larger particles. The supernatant was transferred to an empty sterile tube and was allowed to stand for an additional 15 min. After being transferred to a new sterile tube, the suspension was adjusted to a 0.5 McFarland turbidity standard by visual comparison. One mL of the adjusted suspension was diluted in 4 mL of sterile saline solution.


Table 1. Herbarium data of plants

To prepare *M. tuberculosis* inoculum using a BACTEC MGIT tube with positive growth, the positive tubes were used beginning from the day after the sample first became positive (day-1 positive), up to and including the fifth day (day-5 positive). The positive tubes that were older than five days were subcultured into fresh growth medium. Tubes that were day-1 and day-2 positive were used in the inoculation procedure for the susceptibility tests. The tubes that were between day-3 and day-5 positive were diluted using 1 mL of the positive broth and 4 mL of sterile saline solution; the 5 mL diluted suspension samples were used for the inoculation procedures.

#### **2.8 Antimycobacterial activity test**

314 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

min), carnosic acid (Rt. 8.55 min), chlorogenic acid (Rt 3.59 min), rosmarinic acid (Rt 3.97 min), apigenin 7-glucoside (Rt 3.89 min), oleuropein (Rt 3.969 min), amentoflavone (Rt 5.16 min), naringin (Rt 3.83 min), rutin hydrate (Rt 3.69 min), hesperidin (Rt 3.85 min). Calibration concentrations were 1,4,5 and 20 ppm except one, apigenin 7-glucoside, was 0.9,

The extracts were screened against four strain, *M. tuberculosis* H37Ra (ATCC 25177), *M. tuberculosis* H37Rv (ATCC 25618) and two-positive *M. tuberculosis* isolates obtained from

Bacterial suspensions of *M. tuberculosis* were prepared either from Lowenstein–Jensen slants or from complete 7H9 broth cultures. To prepare an inoculum that was less than 15 days old from a culture grown on Lowenstein-Jensen medium, a suspension was prepared in Middlebrook 7H9 broth. The turbidity of the suspension was adjusted to a 1.0 McFarland standard. The suspension was vortexed for several minutes and was allowed to stand for 20 min for the initial settling of larger particles. The supernatant was transferred to an empty sterile tube and was allowed to stand for an additional 15 min. After being transferred to a new sterile tube, the suspension was adjusted to a 0.5 McFarland turbidity standard by visual comparison. One mL of the adjusted suspension was diluted in 4 mL of sterile saline

(m)

Erzincan 1230 15.Jul.2009 FS 1605

Savastepe 200 02.Jul.2009 FS1561

village 980 20.Sep.2010 FS1562

Kazdagi, 350 23.Jun.2009 FS1603

Kazdagi, 1260 17.Jul.2009 FS1604

forest 850 03.Aug.2009 FS1558

Collection Time

Herbarium Number

1.8, 4.5, 9, and 18 ppm and injection volume was 5 µL for all standards.

patient from hospital, for antibacterial activity.

**2.7 Preparation of** *Mycobacterium tuberculosis* **inocula** 

(Lamiaceae) Locality Altitude

2 *Origanum sipyleum* L. Between Balkesir-

<sup>7</sup>*Stachys cretica* L*.* Balikesir-Edremit,

<sup>9</sup>*Thymus syriacus* Boiss. Gaziantep-Burc

Between Elazig-

3 *Salvia viridis* L. Balikesir-Cagis 160 02. Jun.2010 FS1560

Adiyaman-Yazibaş

Balikesir-Edremit,

Boiss&Bal. Van, Gurpinar 1100 26.Jun.2009 FS 1559

C.Koch. Bursa, Mezitler 860 08.Jul.2009 FS1602

Boiss&Bal.*(endemic)* Antalya, Belek 1000 12.Jul.2010 FS1556

**2.6 Organisms** 

solution.

No Genus species authority

(Hand.-Mazz.) Ietswaart.

<sup>5</sup>*Satureja boissieri* Hausskn.

<sup>1</sup>*Origanum acutidens* 

<sup>4</sup>*Salvia microstegia*

<sup>6</sup>*Stachys byzantine* 

<sup>8</sup>*Stachys cretica* subsp. *smyrnaea* Rech*.*fil.

Table 1. Herbarium data of plants

<sup>10</sup>*Thymus cilicicus* 

ex Boiss.

Antimycobacterial bioassay was performed using the Microplate Alamar Blue Assay (MABA) method (Collins and Franzblau, 1997). MIC was recorded as the lowest drug concentration that prevented to turn blue to pink colour by adding Alamar blue. MBC was also recorded the minimum extract concentration that do not cause any color changing in cultures reincubated in fresh medium.

#### **2.8.1 Determination of Minimal Inhibitory Concentrations (MICs) for** *Mycobacterium tuberculosis*

Microplates were inoculated with the bacterial suspension (20 μL per well except for the negative control wells) and incubated at 37 °C for 6 days. Alamar blue (15 μL, Trek Diagnostic system) was then added to the bacterial growth control wells (without extract) and the microplates were incubated at 37 °C for an additional 24 hours. If the dye turned from blue to pink, (indicating positive bacterial growth) then Alamar blue solution was added to the other wells to determine the MIC values. All tests were performed in triplicate.

## **2.8.2 Determination of mycobactericidal activity**

All the extracts prepared from aerial parts of plants, the herbarium data of these species shown in Table 1, were analyzed by LC-MS. The quantity of chemicals in the methanol extracts are given in Table 2. Chromatograms of phenols in all extracts were compared to chromatograms of standards (Figs. 1–3).

The plant extracts described above were used in mycobactericidal activity tests. Two-fold dilution series in triplicate sets of parallel microplate wells were used for each extract. To determine the minimum bactericide concentrations (MBCs), fresh Middlebrook 7H9 culture broth (185 μL) was transferred to each well. A fifteen microliter of an Mycobacterial suspension, from MIC concentration and higher concentration wells obtained from the MIC test described above was added to each well, in order to determine the minimum bactericide concentration (MBC).

Two microplate wells were used as positive and negative controls, and each test was repeated in triplicate. For the negative controls, 200 mL of fresh broth (Middlebrook 7H9 culture medium and OADC) was used. For positive controls, including 185 μL and inoculums from former positive control wells (15 μL) was used. After 24 hours of incubation and colour development using the Alamar blue solution, MBCs were recorded as the minimum extract concentration that did not cause any colour change in cultures when reincubated in fresh medium.

Antimycobacterial Activity Some Different Lamiaceae

hesperidin

rutin hydrate 20; hesperidin

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 317

Fig. 1. ESI-MS Spectra of standard phenolics, 1; trans-cinnamic acid 2; p-coumaric acid 3; vanillic acid 4; gallic acid 5; caffeic acid 6; ferulic acid 7; apigenin 8; naringenin 9; luteolin 10; epicatechin 11; quercetin 12; carnosic acid 13; chlorogenic acid 14; rosmarinic acid 15; apigenin 7-glucoside 16; amentoflavone 17; oleuropein 18; naringin 19; rutin hydrate 20;

Fig. 2. ESI-TIC SIM chromatogram of standard phenolics, 1; trans-cinnamic acid 2; pcoumaric acid 3; vanillic acid 4; gallic acid 5; caffeic acid 6; ferulic acid 7; apigenin 8; naringenin 9; luteolin 10; epicatechin 11; quercetin 12; carnosic acid 13; chlorogenic acid 14; rosmarinic acid 15; apigenin 7-glucoside 16; amentoflavone 17; oleuropein 18; naringin 19;

## **3. Results**

## **3.1 Phenolics determined by LC-MS analyses**

The ten samples and selected standards were analyzed by MS in ESI negative ion mode. Scan mode is also used. In this method, trans-cinnamic acid, p-coumaric acid, vanillic acid, gallic acid, caffeic acid, ferulic acid, apigenin, naringenin, luteolin, epicatechin, quercetin, carnosic acid, chologenic acid, rosmarinic acid, apigenin 7-glucoside, amentoflavone, oleuropein, naringin, rutin hydrate and hesperidin were chosen as standard phenolics to determine the phenolic structures of the samples according to ionization response in ESI mass spectrometry and chromatographic retention time.

Ion profile of negative ion electrospray LC/MS analysis experimental conditions are given above, from plants CL, EA and ME extracts is shown Fig. 1-2 and Table 2. Phenolics of samples were identified by comparing standard phenolic data such as retention times, main ions observed under fragmentation voltage of 80 Volt.


Table 2. LS-MS characteristics of phenolic compounds

The major phenolic compounds of *T. cilicicus* CL extract were rutin hydrate and naringenin; for EA extract, rosmarinic acid and apigenin; and for ME extract, rosmarinic acid, oleropein, and apigenin.

The highest rosmarinic acid level within all plants were determined in *S. viridis* for CL extracts; in *S. boissieri* and *T. cilicicus* for EA extracts; *O. sipyleum S. byzantine* and *S. boissieri* for ME extracts.

The ten samples and selected standards were analyzed by MS in ESI negative ion mode. Scan mode is also used. In this method, trans-cinnamic acid, p-coumaric acid, vanillic acid, gallic acid, caffeic acid, ferulic acid, apigenin, naringenin, luteolin, epicatechin, quercetin, carnosic acid, chologenic acid, rosmarinic acid, apigenin 7-glucoside, amentoflavone, oleuropein, naringin, rutin hydrate and hesperidin were chosen as standard phenolics to determine the phenolic structures of the samples according to ionization response in ESI

Ion profile of negative ion electrospray LC/MS analysis experimental conditions are given above, from plants CL, EA and ME extracts is shown Fig. 1-2 and Table 2. Phenolics of samples were identified by comparing standard phenolic data such as retention times, main

No Phenolics Rt min [M-H]- Fragment ions 1 *trans*-Cinnamic acid 4,984 147 80 2 ρ-Coumaric acid 3,95 163 80 3 4-Hydroxy-3-metoxybenzoic acid (vanillic acid) 3,747 167 80 4 Gallic acid monohyrate 1,893 169 80 5 Caffeic acid 3,724 179 80 6 Ferulic acid 3,991 193 80 7 Apigenin 4,831 269 80 8 (+)-Naringenin 4,859 271 80 9 Luteolin 4,433 285 80 10 (-)-Epicatechin 3,675 289 80 11 Quercetin 4,427 301 80 12 Carnosic acid 8,555 331 80 13 Chlorogenic acid 3,597 353 80 14 Rosmarinic acid 3,971 359 80 15 Apigenin 7-glucoside 3,896 431 80 16 Amentoflavone 5,169 537 80 17 Oleuropein 3,969 539 80 18 Naringin 3,834 579 80 19 Rutin hydrate 3,699 609 80 20 Hesperidin 3,853 609 80

The major phenolic compounds of *T. cilicicus* CL extract were rutin hydrate and naringenin; for EA extract, rosmarinic acid and apigenin; and for ME extract, rosmarinic acid, oleropein,

The highest rosmarinic acid level within all plants were determined in *S. viridis* for CL extracts; in *S. boissieri* and *T. cilicicus* for EA extracts; *O. sipyleum S. byzantine* and *S. boissieri*

**3. Results** 

**3.1 Phenolics determined by LC-MS analyses** 

mass spectrometry and chromatographic retention time.

ions observed under fragmentation voltage of 80 Volt.

Table 2. LS-MS characteristics of phenolic compounds

and apigenin.

for ME extracts.

Fig. 1. ESI-MS Spectra of standard phenolics, 1; trans-cinnamic acid 2; p-coumaric acid 3; vanillic acid 4; gallic acid 5; caffeic acid 6; ferulic acid 7; apigenin 8; naringenin 9; luteolin 10; epicatechin 11; quercetin 12; carnosic acid 13; chlorogenic acid 14; rosmarinic acid 15; apigenin 7-glucoside 16; amentoflavone 17; oleuropein 18; naringin 19; rutin hydrate 20; hesperidin

Fig. 2. ESI-TIC SIM chromatogram of standard phenolics, 1; trans-cinnamic acid 2; pcoumaric acid 3; vanillic acid 4; gallic acid 5; caffeic acid 6; ferulic acid 7; apigenin 8; naringenin 9; luteolin 10; epicatechin 11; quercetin 12; carnosic acid 13; chlorogenic acid 14; rosmarinic acid 15; apigenin 7-glucoside 16; amentoflavone 17; oleuropein 18; naringin 19; rutin hydrate 20; hesperidin

Antimycobacterial Activity Some Different Lamiaceae

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 319

D

E Fig. 3*.* ESI-TIC SIM chromatogram of *O.sipyleum* A) EA extract, B) ME extract; *S. boissieri* C)

The major phenolic compounds for *T. syriacus* were rutin hydrate and naringenin for CL extract; rosmarinic acid, apigenin naringenin, and vanillic acid for EA extract; rosmarinic

The major phenolic compounds of *O. acutidens* determined by LC-MS analyses were rutin hydrate for the CL extracts; rosmarinic acid and oleuropein for the EA extracts, rosmarinic acid; and vanillic acid for the ME extracts. The major phenolics of *O. sipyleum* were rutin hydrate for CL extracts; rosmarinic acid and vanillic acid for EA and ME extracts. The major phenolics of CL extracts of *S. viridis* were rosmarinic acid and rutin hydrate; for EA extracts, oleuropein followed by rosmarinic acid; for ME extracts, rosmarinic acid, chlorogenic acid

The major phenolic compounds for *S. microstegia* were rutin hydrate for CL extracts; apigenin, luteolin, and rosmarinic acid for EA extract; and rosmarinic acid, apigenin and luteolin for ME extracts. In *S. boissieri*, the major phenolics for CL extracts were apigenin and naringenin; for EA extracts, rosmarinic acid, naringenin and hesperidin; for ME extracts, rosmarinic acid and hesperidin (Fig 3). The major phenolic compounds in the CL extracts of

CL extract D) EA etract and E) ME extract. (Not all chromatograms are included).

acid, apigenin, luteolin, and oleropein for ME extract.

and hesperidin.

A

B

C

#### Antimycobacterial Activity Some Different Lamiaceae Plant Extracts Containing Flavonoids and Other Phenolic Compounds 319

318 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

A

B

C

D

E

Fig. 3*.* ESI-TIC SIM chromatogram of *O.sipyleum* A) EA extract, B) ME extract; *S. boissieri* C) CL extract D) EA etract and E) ME extract. (Not all chromatograms are included).

The major phenolic compounds for *T. syriacus* were rutin hydrate and naringenin for CL extract; rosmarinic acid, apigenin naringenin, and vanillic acid for EA extract; rosmarinic acid, apigenin, luteolin, and oleropein for ME extract.

The major phenolic compounds of *O. acutidens* determined by LC-MS analyses were rutin hydrate for the CL extracts; rosmarinic acid and oleuropein for the EA extracts, rosmarinic acid; and vanillic acid for the ME extracts. The major phenolics of *O. sipyleum* were rutin hydrate for CL extracts; rosmarinic acid and vanillic acid for EA and ME extracts. The major phenolics of CL extracts of *S. viridis* were rosmarinic acid and rutin hydrate; for EA extracts, oleuropein followed by rosmarinic acid; for ME extracts, rosmarinic acid, chlorogenic acid and hesperidin.

The major phenolic compounds for *S. microstegia* were rutin hydrate for CL extracts; apigenin, luteolin, and rosmarinic acid for EA extract; and rosmarinic acid, apigenin and luteolin for ME extracts. In *S. boissieri*, the major phenolics for CL extracts were apigenin and naringenin; for EA extracts, rosmarinic acid, naringenin and hesperidin; for ME extracts, rosmarinic acid and hesperidin (Fig 3). The major phenolic compounds in the CL extracts of

Antimycobacterial Activity Some Different Lamiaceae

Pt: Plants; Exts: Extracts; CL: Chloroform; EA: Ethyl Acetat; ME: Methanol; 1; Rosmarinic Acid 2; Naringin 3; Quercetin 4; Epicatechin 5; Rutin Hydrate 6; Caffeic Acid 7; Gallic Acid 8; Trans-cinnamic Acid 9; ρ-coumaric acid 10; Vanillic Acid 11; Ferulic Acid 12; Naringenin 13; Chlorogenic Acid 14; Luteolin 15; Apigenin 7-glucoside 16; Hesperidin 17; Oleuropein 18; Carnosic Acid 19; Amentoflavone 20; Apigenin

Lamiaceae species.

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 321

Table 3. Chemical concentrations in chloroform, ethyl acetat and methanol extracts of


Table 3. Chemical concentrations in chloroform, ethyl acetat and methanol extracts of Lamiaceae species.

320 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Antimycobacterial Activity Some Different Lamiaceae

*Origanum acutidens*

*O. sipyleum*

*Salvia viridis*

*S. microstegia*

*S. boissieri*

*S. cretica*

*Stachys byzantine*

*Thymus syriacus*

*T. cilicicus*

*S. cretica* subsp. *smyrnaea*

MIC:( mg/mL); MBC: ( mg/mL;). n.t: not tested.

Table 4. Antibacterial activity of extracts of the plants as MIC (mg/mL) and MBC

H37Rv (ATCC 25618) obtained by MABA (*Microplate Alamar blue* assay) method.

susceptibility test results against M. tuberculosis H37Ra (ATCC 25177) and *M. tuberculosis* 

same effect at MIC and MBC 3.1 on *M. tuberculosis* H37 Rv.

ranged between 6.3-12.5 and 6.3-25 mg/mL, respectively (Table 4).

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 323

effective at MIC and MBC 6.3 mg/mL against two TB-positive isolates; *T. cilicus* showed the

Of the ME extracts, the most effective against *M. tuberculosis* H37 Ra was *T. syriacus* (MIC and MBC 3.1 mg/mL). *Stachys byzantine* also showed considerable efficacy at MIC 3.1 mg/mL against TB-positive isolates1. Among the other extracts, MIC and MBC values

Plants **Extracts H37Ra H37***Rv* **Isolate1 Isolate2** 

MIC MBC MIC MBC MIC MBC MIC MBC

Cl 0.4 6.3 12.5 12.5 12.5 25 6.3 6.3 Ea 6.3 6.3 6.3 6.3 3.1 6.3 3.1 6.3 Me 6.3 6.3 25 25 3.1 12.5 6.3 12.5

Cl 3.1 12.5 25 25 12.5 25 6.3 12.5 Ea 1.6 3.1 25 25 6.3 25 6.3 25 Me 12.5 12.5 25 25 6.3 12.5 6.3 6.3

Cl 6.3 12.5 12.5 12.5 n.t n.t n.t n.t Ea 1.6 3.1 12.5 12.5 6.3 12.5 6.3 12.5 Me 12.5 12.5 25 25 6.3 25 6.3 25

Cl 0.4 12.5 12.5 12.5 12.5 25 6.3 12.5 Ea 6.3 12.5 25 25 3.1 12.5 3.1 12.5 Me 12.5 12.5 12.5 25 6.3 25 6.3 25

Cl 0.8 6.3 0.4 0.8 0.8 0.8 0.8 0.8 Ea 1.6 3.1 12.5 12.5 3.1 3.1 3.1 3.1 Me 12.5 12.5 12.5 12.5 6.3 12.5 6.3 12.5

Cl 0.8 12.5 25 25 12.5 25 6.3 6.3 Ea 12.5 12.5 12.5 12.5 3.1 6.3 6.3 12.5 Me 12.5 12.5 25 25 3.1 12.5 6.3 25

Cl 0.8 12.5 6.3 12.5 12.5 12.5 6.3 12.5 Ea 1.6 12.5 6.3 12.5 3.1 12.5 3.1 12.5 Me 12.5 12.5 12.5 12.5 6.3 25 6.3 25

Cl 6.3 12.5 12.5 12.5 3.1 3.1 6.3 12.5 Ea 6.3 12.5 12.5 12.5 3.1 12.5 3.1 12.5 Me 25 25 12.5 12.5 3.1 25 3.1 25

Cl 0.4 12.5 6.3 12.5 6.3 6.3 3.1 6.3 Ea 0.8 1.6 3.1 3.1 3.1 12.5 3.1 12.5 Me 3.1 3.1 50 50 6.3 12.5 6.3 25

Cl 0.8 12.5 6.3 25 6.3 25 6.3 12.5 Ea 3.1 6.3 3.1 6.3 3.1 25 3.1 25 Me 12.5 12.5 12.5 12.5 6.3 25 6.3 25

*S. byzantina* were rutin hydrate; in the EA extract, apigenin, luteolin and rosmarinic acid; and in the ME extract, rosmarinic acid, hesperidin and apigenin.

In *S. cretica*, the major phenolics in the CL extracts were trans-cinnamic acid and vanillic acid; oleuropein, vanillic acid and rosmarinic acid for EA extract; and chlorogenic acid and rosmarinic acid for ME extract. In *S. smyrnaea*, the major phenolics were rosmarinic acid and rutin hydrate for CL extracts; vanillic acid and chlorogenic acid for EA extract; chlorogenic acid and hesperidin for ME extracts.

The highest rutin hydrate contents were determined in *O. sipyleum* and *S. viridis* for CL extracts; *T. cilicicus, S. viridis, and S. boissieri* for EA extracts*; S. cretica* subsp*. smyrnaea, S. byzantina*, and *T. syriacus* for ME extracts.

Gallic acid was determined only in methanol extracts of *S. viridis*. Carnosic acid was also found in CL extract of *S. boissieri*. Only the EA extracts of *S. microstegia*, S*. byzantina, T. cilicicus* and *T. striacus* included the highest level of apigenin.

Trans-cinnamic acid was found in extracts of four plants (*O. acutidens*, S*. byzantina*, and *S. cretica* subsp. *smyrnaea*). Quercetin and amentoflavone were not found. The highest level of chlorogenic acid was found in ME extracts of *S. cretica* subsp. *smyrnaea*, *S. cretica*, and *S. viridis*. Luteolin occurred mostly in EA and ME extracts in *S. microstegia*, S*. byzantina*, and *T. cilicicus*. The highest hesperidin level was found in *S. boissieri* ME extract and it follows *S. byzantina* ME extracts; In addition, ME extracts of *S. viridis* and *T*. *cilicicus* also included high levels of hesperidin. The highest oleuropein content was determined in ME extracts of *T. cilicicus*, followed by *T. syriacus* and *S. boissieri*. Within EA extracts*, S. viridis* and *O. acutidens* had the highest level of oleuropein.

#### **3.2 Antimycobacterial activities, MICs & MBCs**

The results were evaluated according to the literature. Extracts were tested against four mycobacteria strains (*M. tuberculosis* H37Ra, *M. tuberculosis* H37Rv, and two-positive *M. tuberculosis* isolates) obtained from hospital patients, to determine the MIC and MBC using the micro dilution method (MABA) against reference strains.

All plant extracts showed antimycobacterial activity (Table 4). Within all CL extracts, *O. acutidens, S. microstegia,* and *T. syriacus* exhibited the lowest MIC value of 0.4 mg/mL against *M. tuberculosis* H37 Ra. The lowest MBC value was 6.3 mg/mL for *O. acutidens* and *S. boissieri.* The MBC value for the rest of species was 12.5 mg/mL.

The MIC value of CL extracts against *M. tuberculosis* H37 Rv was 0.4 mg/mL for *S. boissieri*, followed by *S. cretica, T. syriacus,* and *T. cilicus* at MIC 6.3 mg/mL. Although all CL extracts showed bactericidal activity against *M. tuberculosis* H37 Rv, the prominent MBC values are 0.8 mg/mL for *S. boissieri* and 3.1 mg/mL for *T. syriacus*. For TB-positive isolates1, the featured results were 0.8 mg/mL MIC and MBC for *S. boissieri* and 3.1 mg/mL MIC and MBC for *S. cretica* subsp. *smyrnaea. S. boissieri* was also effective at the concentration 0.8 mg/mL as MBC.

In the EA extracts, the most prominent efficacy was observed for *T. syriacus* at MIC 0.8 mg/mL; MBC 1.6 mg/mL for *T. syriacus* against *M. tuberculosis* H37 Ra. *S. boissieri* is also

*S. byzantina* were rutin hydrate; in the EA extract, apigenin, luteolin and rosmarinic acid;

In *S. cretica*, the major phenolics in the CL extracts were trans-cinnamic acid and vanillic acid; oleuropein, vanillic acid and rosmarinic acid for EA extract; and chlorogenic acid and rosmarinic acid for ME extract. In *S. smyrnaea*, the major phenolics were rosmarinic acid and rutin hydrate for CL extracts; vanillic acid and chlorogenic acid for EA extract; chlorogenic

The highest rutin hydrate contents were determined in *O. sipyleum* and *S. viridis* for CL extracts; *T. cilicicus, S. viridis, and S. boissieri* for EA extracts*; S. cretica* subsp*. smyrnaea, S.* 

Gallic acid was determined only in methanol extracts of *S. viridis*. Carnosic acid was also found in CL extract of *S. boissieri*. Only the EA extracts of *S. microstegia*, S*. byzantina, T.* 

Trans-cinnamic acid was found in extracts of four plants (*O. acutidens*, S*. byzantina*, and *S. cretica* subsp. *smyrnaea*). Quercetin and amentoflavone were not found. The highest level of chlorogenic acid was found in ME extracts of *S. cretica* subsp. *smyrnaea*, *S. cretica*, and *S. viridis*. Luteolin occurred mostly in EA and ME extracts in *S. microstegia*, S*. byzantina*, and *T. cilicicus*. The highest hesperidin level was found in *S. boissieri* ME extract and it follows *S. byzantina* ME extracts; In addition, ME extracts of *S. viridis* and *T*. *cilicicus* also included high levels of hesperidin. The highest oleuropein content was determined in ME extracts of *T. cilicicus*, followed by *T. syriacus* and *S. boissieri*. Within EA extracts*, S. viridis* and *O. acutidens*

The results were evaluated according to the literature. Extracts were tested against four mycobacteria strains (*M. tuberculosis* H37Ra, *M. tuberculosis* H37Rv, and two-positive *M. tuberculosis* isolates) obtained from hospital patients, to determine the MIC and MBC using

All plant extracts showed antimycobacterial activity (Table 4). Within all CL extracts, *O. acutidens, S. microstegia,* and *T. syriacus* exhibited the lowest MIC value of 0.4 mg/mL against *M. tuberculosis* H37 Ra. The lowest MBC value was 6.3 mg/mL for *O. acutidens* and *S.* 

The MIC value of CL extracts against *M. tuberculosis* H37 Rv was 0.4 mg/mL for *S. boissieri*, followed by *S. cretica, T. syriacus,* and *T. cilicus* at MIC 6.3 mg/mL. Although all CL extracts showed bactericidal activity against *M. tuberculosis* H37 Rv, the prominent MBC values are 0.8 mg/mL for *S. boissieri* and 3.1 mg/mL for *T. syriacus*. For TB-positive isolates1, the featured results were 0.8 mg/mL MIC and MBC for *S. boissieri* and 3.1 mg/mL MIC and MBC for *S. cretica* subsp. *smyrnaea. S. boissieri* was also effective at the concentration 0.8

In the EA extracts, the most prominent efficacy was observed for *T. syriacus* at MIC 0.8 mg/mL; MBC 1.6 mg/mL for *T. syriacus* against *M. tuberculosis* H37 Ra. *S. boissieri* is also

and in the ME extract, rosmarinic acid, hesperidin and apigenin.

*cilicicus* and *T. striacus* included the highest level of apigenin.

acid and hesperidin for ME extracts.

had the highest level of oleuropein.

mg/mL as MBC.

**3.2 Antimycobacterial activities, MICs & MBCs** 

the micro dilution method (MABA) against reference strains.

*boissieri.* The MBC value for the rest of species was 12.5 mg/mL.

*byzantina*, and *T. syriacus* for ME extracts.

effective at MIC and MBC 6.3 mg/mL against two TB-positive isolates; *T. cilicus* showed the same effect at MIC and MBC 3.1 on *M. tuberculosis* H37 Rv.

Of the ME extracts, the most effective against *M. tuberculosis* H37 Ra was *T. syriacus* (MIC and MBC 3.1 mg/mL). *Stachys byzantine* also showed considerable efficacy at MIC 3.1 mg/mL against TB-positive isolates1. Among the other extracts, MIC and MBC values ranged between 6.3-12.5 and 6.3-25 mg/mL, respectively (Table 4).


MIC:( mg/mL); MBC: ( mg/mL;). n.t: not tested.

Table 4. Antibacterial activity of extracts of the plants as MIC (mg/mL) and MBC susceptibility test results against M. tuberculosis H37Ra (ATCC 25177) and *M. tuberculosis*  H37Rv (ATCC 25618) obtained by MABA (*Microplate Alamar blue* assay) method.

Antimycobacterial Activity Some Different Lamiaceae

system.

Gram-negative cell wall barrier.

ethnomedicinal knowledge on plants.

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 325

*T. spicata* var. *spicata* was more effective (MIC 196 µg/ml) than *O. minutiflorum* (MIC 392 µg/ml). They suggested that a high quantity of rosmarinic acid might be one of the responsible constituent for the observed antimycobacterial activity. Gordien et al. (2009) studied two terpenes, sesquiterpene and longifolene; and two diterpenes, totarol and transcommunic acid, obtained from the aerial parts and roots of *Juniperus communis*. They reported that totarol showed the highest activity against *Mycobacterium tuberculosis* H37Rv and that longifolene and totarol exhibited the most activity against rifampicin-resistant variants. These results supported the ethnomedicinal use of this species as a traditional anti-TB remedy. Kuete et al. (2010) investigated the antimycobacterial activity of five flavonoids (isobachalcone, kanzanol C, 4-hydroxylonchocarpin, stipulin, amentoflavone) and determined their effects on preventing the growth of mycobacteria with MIC < 10 µg/ml on *M. tuberculosis*. In addition, isobachalcone and stipulin showed total inhibition of *M. tuberculosis* strain H37Rv. Bernard et al. (1997) mentioned that rutin showed antibacterial activity on *E. coli* by inhibited topoisomerase IV-dependent decatenation activity and caused *E. coli* topoisomerase IV which is essential for cell survival, dependent DNA cleavage (Bernard et al., 1997; Normark et al., 1969; Cushnie& Lamb., 2005). Huang et al. (2008) indicated that evidence that vanillic acid might be helpful to prevent of the development of the development of diabetic neuropathy by blocking the methylglyoxal-mediated glycation

Mandalari et al. (2007) also reported that, pair-wise combinations of eriodictyol, naringenin and hesperidin showed both synergistic and indifferent interactions that were dependent on the test indicator organism and their cell wall structure. Parekh and Chanda (2007) reported that the crude methanol extract of *Woodfordia fruticosa* contains certain constituents, such as tannins, with significant antibacterial properties, which enables the extract to overcome the

Kamatou et al. (2007) studied 16 South African *Salvia* species that are used in traditional medicine to treat microbial infection. They identified three species, *S. verbenaca, S. radula* and *S. dolomitica*, which exhibited MIC value at 0.10 mg/mL and which also showed antibacterial activity. Green et al. (2010) reported on the activities of acetone extracts of four plants, while *Berchemia discolor* showed efficacy at MIC 12. 5µg/mL, on H37Ra and 10.5µg/mL on the clinical isolate; the others (*Bridelia micrantha*, *Warbugia salutaris*, and *Terminalia sericea*) showed efficacy at 25µg/mL on both H37Ra and clinical isolate. The authors validated that these plants include mycobactericidal compounds that are effective against multidrug-resistant *M. tuberculosis.* Graham et al. (2003) presented an antimycobacterial evaluation of 216 species of Peruvian plants (in 63 families). Dichloromethane extracts from slightly more than half of the samples tested showed MIC value at 50 μg/ml concentration against *M. tuberculosis.* Billo et al. (2005) reported that methanolic extract of *Amborella trichopoda* fruits shows MIC value between 1 and 2.5 µg/ml,

which was better than pyrazynamide and ethambutol in the same conditions.

Fabryet et al. (1998) reported that solvent extracts of plants with MIC values less than 8 mg/mL may be considered as antimicrobially effective. Gautam et al., (2007), shows that extracts of plant species from wide range of families and genera have exhibited significant in vitro antimycobacterial activities and this efficacy is interestingly compatible with the

## **4. Discussion**

Lamiaceae plant extracts prepared by using different plant parts such as bark, stem, root, leaves, and fruits used in many biological activity studies. The extracts have been found to have antibacterial activity (Alma et al., 2003; Amanlou et al., 2004; Digrak et al., 2001; Bozin et al., 2006; Karaman et al., 2001), antifungal activity (Bouchra et al., 2003; Askun et al., 2008; Gulluce et al., 2003; Guynot et al., 2003; Souza et al., 2005), antimycobacterial activity (Ulubelen et al., 1997; Askun et al., 2009), antioxidant activity (Alma et al., 2003; Bozin et al., 2006; Mosaffa et al., 2006; Gulluce et al., 2003) and anti-inflammatory activity (Alcar´az et al., 1989; Jim´enez et al., 1986). Inhibitory effects of oregano components on some foodborne fungi were reported (Akgul & Kivanc, 1988). Askun et al. (2009) indicated that *Origanum minutiflorum* and *Thymbra spicata* methanol extracts showed antimycobacterial activity against *M. tuberculosis. T. spicata* var*. spicata* showed greater antimycobacterial efficacy (at MIC 196 µg/ml) than *O. minutiflorum* (MIC 392 µg/ml). They stated that a high quantity of rosmarinic acid might be responsible for antimycobacterial activity.

Recently, investigations of plant extracts are attracting great attentions due to their antibacterial properties (Payne et al., 2007; Rukayadi et al., 2009; Guzman et al., 2010). Previous studies showed that some plant extracts were conciderably effective against *M. tuberculosis*. Lall and Meyer (1999) reported that growth of *M*. *tuberculosis* is inhibited by acetone and water extracts of *Cryptocarya latifolia*, *Euclea natalensis*, *Helichrysum melanacme*, *Nidorella anomala* and *Thymus* v*ulgaris.* They screened these active acetone extracts against H37Rv and a TB strain that was resistant to the drugs isoniazid and rifampicin. They reported that, while some plants (*Croton pseudopulchellus*, *Ekebergia capensis*, *Euclea natalensis*, *Nidorella anomala* and *Polygala myrtifolia)* exhibited MIC at 0.1 mg/mL against H37Rv, others (*Chenopodium ambrosioides*, *Ekebergia capensis*, *Euclea natalensis*, *Helichrysum melanacme*, *Nidorella anomala* and *Polygala myrtifolia)* inhibited the resistant strain at the same MIC value.

Many natural products have attracted much attention as potential antimycobacterial agents (Kinghorn, 2001; Gupta et al., 2010; Guzman et al., 2010). In recent years, there are pleny of researches on phenolics and their biological activities involved in the literature. Phenolic compounds obtained from plant extracts show great variety, with at least 8000 different structures (Bravo, 1998). Estevinho et al. (2008) showed that differences in the profiles of phenolic compounds are dependent of the flora predominance. Chun et al. (2005) reported that high phenolic and antioxidant activity was related to high antimicrobial activity against ulcer-associated *H. pylori*. Cinnamic acid is a naturally occurring phenolic compound that shows antimicrobial activity. Chen et al. (2011) showed that cis-cinnamic acid that was transformed from trans-cinnamic acid showed higher synergistic effect with INH or RIF against tuberculosis than trans-cinnamic acid.

Siedel & Taylor (2004) investigated plants, *Pelargonium reniforme* and *P. sidoides* (Geraniaceae) fractionation of n-hexane extracts against *M. aurum, M. smegmatis, M. fortuitum, M. abscessus* and *M. phlei*. They reported that linoleic acid was the most potent compound (MIC of 2 mg/l) against *M. aurum*. Koysomboon et al. (2006) isolated flavonoids from the stems and roots of the mangrove plant *Derris indica*. They reported antimycobacterial activity at MIC values between 6.25 and 200 µg/mL, except in two of ten known compounds. Askun et al. (2009) indicated that *Origanum minutiflorum* and *Thymbra spicata* var. *spicata* methanol extracts have antimycobacterial activity against *M. tuberculosis.* 

Lamiaceae plant extracts prepared by using different plant parts such as bark, stem, root, leaves, and fruits used in many biological activity studies. The extracts have been found to have antibacterial activity (Alma et al., 2003; Amanlou et al., 2004; Digrak et al., 2001; Bozin et al., 2006; Karaman et al., 2001), antifungal activity (Bouchra et al., 2003; Askun et al., 2008; Gulluce et al., 2003; Guynot et al., 2003; Souza et al., 2005), antimycobacterial activity (Ulubelen et al., 1997; Askun et al., 2009), antioxidant activity (Alma et al., 2003; Bozin et al., 2006; Mosaffa et al., 2006; Gulluce et al., 2003) and anti-inflammatory activity (Alcar´az et al., 1989; Jim´enez et al., 1986). Inhibitory effects of oregano components on some foodborne fungi were reported (Akgul & Kivanc, 1988). Askun et al. (2009) indicated that *Origanum minutiflorum* and *Thymbra spicata* methanol extracts showed antimycobacterial activity against *M. tuberculosis. T. spicata* var*. spicata* showed greater antimycobacterial efficacy (at MIC 196 µg/ml) than *O. minutiflorum* (MIC 392 µg/ml). They stated that a high quantity of

Recently, investigations of plant extracts are attracting great attentions due to their antibacterial properties (Payne et al., 2007; Rukayadi et al., 2009; Guzman et al., 2010). Previous studies showed that some plant extracts were conciderably effective against *M. tuberculosis*. Lall and Meyer (1999) reported that growth of *M*. *tuberculosis* is inhibited by acetone and water extracts of *Cryptocarya latifolia*, *Euclea natalensis*, *Helichrysum melanacme*, *Nidorella anomala* and *Thymus* v*ulgaris.* They screened these active acetone extracts against H37Rv and a TB strain that was resistant to the drugs isoniazid and rifampicin. They reported that, while some plants (*Croton pseudopulchellus*, *Ekebergia capensis*, *Euclea natalensis*, *Nidorella anomala* and *Polygala myrtifolia)* exhibited MIC at 0.1 mg/mL against H37Rv, others (*Chenopodium ambrosioides*, *Ekebergia capensis*, *Euclea natalensis*, *Helichrysum melanacme*, *Nidorella anomala* and *Polygala myrtifolia)* inhibited the resistant strain at the same MIC value. Many natural products have attracted much attention as potential antimycobacterial agents (Kinghorn, 2001; Gupta et al., 2010; Guzman et al., 2010). In recent years, there are pleny of researches on phenolics and their biological activities involved in the literature. Phenolic compounds obtained from plant extracts show great variety, with at least 8000 different structures (Bravo, 1998). Estevinho et al. (2008) showed that differences in the profiles of phenolic compounds are dependent of the flora predominance. Chun et al. (2005) reported that high phenolic and antioxidant activity was related to high antimicrobial activity against ulcer-associated *H. pylori*. Cinnamic acid is a naturally occurring phenolic compound that shows antimicrobial activity. Chen et al. (2011) showed that cis-cinnamic acid that was transformed from trans-cinnamic acid showed higher synergistic effect with INH or RIF

Siedel & Taylor (2004) investigated plants, *Pelargonium reniforme* and *P. sidoides* (Geraniaceae) fractionation of n-hexane extracts against *M. aurum, M. smegmatis, M. fortuitum, M. abscessus* and *M. phlei*. They reported that linoleic acid was the most potent compound (MIC of 2 mg/l) against *M. aurum*. Koysomboon et al. (2006) isolated flavonoids from the stems and roots of the mangrove plant *Derris indica*. They reported antimycobacterial activity at MIC values between 6.25 and 200 µg/mL, except in two of ten known compounds. Askun et al. (2009) indicated that *Origanum minutiflorum* and *Thymbra spicata* var. *spicata* methanol extracts have antimycobacterial activity against *M. tuberculosis.* 

rosmarinic acid might be responsible for antimycobacterial activity.

against tuberculosis than trans-cinnamic acid.

**4. Discussion** 

*T. spicata* var. *spicata* was more effective (MIC 196 µg/ml) than *O. minutiflorum* (MIC 392 µg/ml). They suggested that a high quantity of rosmarinic acid might be one of the responsible constituent for the observed antimycobacterial activity. Gordien et al. (2009) studied two terpenes, sesquiterpene and longifolene; and two diterpenes, totarol and transcommunic acid, obtained from the aerial parts and roots of *Juniperus communis*. They reported that totarol showed the highest activity against *Mycobacterium tuberculosis* H37Rv and that longifolene and totarol exhibited the most activity against rifampicin-resistant variants. These results supported the ethnomedicinal use of this species as a traditional anti-TB remedy. Kuete et al. (2010) investigated the antimycobacterial activity of five flavonoids (isobachalcone, kanzanol C, 4-hydroxylonchocarpin, stipulin, amentoflavone) and determined their effects on preventing the growth of mycobacteria with MIC < 10 µg/ml on *M. tuberculosis*. In addition, isobachalcone and stipulin showed total inhibition of *M. tuberculosis* strain H37Rv. Bernard et al. (1997) mentioned that rutin showed antibacterial activity on *E. coli* by inhibited topoisomerase IV-dependent decatenation activity and caused *E. coli* topoisomerase IV which is essential for cell survival, dependent DNA cleavage (Bernard et al., 1997; Normark et al., 1969; Cushnie& Lamb., 2005). Huang et al. (2008) indicated that evidence that vanillic acid might be helpful to prevent of the development of the development of diabetic neuropathy by blocking the methylglyoxal-mediated glycation system.

Mandalari et al. (2007) also reported that, pair-wise combinations of eriodictyol, naringenin and hesperidin showed both synergistic and indifferent interactions that were dependent on the test indicator organism and their cell wall structure. Parekh and Chanda (2007) reported that the crude methanol extract of *Woodfordia fruticosa* contains certain constituents, such as tannins, with significant antibacterial properties, which enables the extract to overcome the Gram-negative cell wall barrier.

Kamatou et al. (2007) studied 16 South African *Salvia* species that are used in traditional medicine to treat microbial infection. They identified three species, *S. verbenaca, S. radula* and *S. dolomitica*, which exhibited MIC value at 0.10 mg/mL and which also showed antibacterial activity. Green et al. (2010) reported on the activities of acetone extracts of four plants, while *Berchemia discolor* showed efficacy at MIC 12. 5µg/mL, on H37Ra and 10.5µg/mL on the clinical isolate; the others (*Bridelia micrantha*, *Warbugia salutaris*, and *Terminalia sericea*) showed efficacy at 25µg/mL on both H37Ra and clinical isolate. The authors validated that these plants include mycobactericidal compounds that are effective against multidrug-resistant *M. tuberculosis.* Graham et al. (2003) presented an antimycobacterial evaluation of 216 species of Peruvian plants (in 63 families). Dichloromethane extracts from slightly more than half of the samples tested showed MIC value at 50 μg/ml concentration against *M. tuberculosis.* Billo et al. (2005) reported that methanolic extract of *Amborella trichopoda* fruits shows MIC value between 1 and 2.5 µg/ml, which was better than pyrazynamide and ethambutol in the same conditions.

Fabryet et al. (1998) reported that solvent extracts of plants with MIC values less than 8 mg/mL may be considered as antimicrobially effective. Gautam et al., (2007), shows that extracts of plant species from wide range of families and genera have exhibited significant in vitro antimycobacterial activities and this efficacy is interestingly compatible with the ethnomedicinal knowledge on plants.

Antimycobacterial Activity Some Different Lamiaceae

*japonica*. Yaoxue Tongbao 15, 39.

1032, ISSN 0031-9422.

ISSN 0021-8561.

0163-3864.

3719.

protease and reverse transcriptase.

*M. tuberculosis* .

**7. References** 

Plant Extracts Containing Flavonoids and Other Phenolic Compounds 327

These strong complexes might disrupt the metabolism of organism (Havsteen, 2002). Flavonoids are also known to have mutagenic and antitumor activities (Hodec et al., 2002; Havsteen, 2002). Quercetin affects bacteria by inhibiting the catalytic activity of DNA topoisomerase I and II (Constantinou et al., 1995; Hodec et al., 2002). Quercetin was also reported by Xu et al. (2000) and Spedding et al. (1989) to have inhibitory effects on HIV1-

It is imperative to investigate the use of new, cheaper and efficient compounds to control *Mycobacteria tuberculosis*. Recent studies have examined plants and the effectiveness of their different types of extracts on *M. tuberculosis*. Advanced research into the structure and activity relationships among naturally occurring flavonoids will yield greater understanding of their pharmacokinetics and effects on mycobacteria metabolism according to their structure. It is of great importance to determine the mechanisms of action of flavonoids on

Huang, PH., Chen, WS., Hu, Y. (1980). Studies on antituberculosis constituents from *Ardisia* 

Okunade, AL., Elvin-Lewis, MPF. & Lewis, WH. (2004) Natural antimycobacterial

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Antunes, ML., Aleixo-Dias, J., Antunes, AF., Pereira, MF., Raymundo, E., & Rodrigues, MF.

Askun, T., Tumen, G., Satil, F., & Kilic, T. (2008). Effects of Some Lamiaceae Species

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Lechner et al. (2008) showed that myricetin was the most efficient intensifier of INH susceptibility in all tested strains by decreasing the MIC value of INH by as much as 64-fold; the second most effective compound was quercetin. Huang et al. (1980) tested two benzenoid compounds isolated from *Ardisia japonica* in-vivo on 201 patients infected with *M. tuberculosis* (Okunade et al., 2004). They reported that both compounds showed over 80% efficacy.

## **5. Acknowledgments**

The authors are grateful to TUBITAK. This research was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK), TBAG (Research Grant No. 104T336). In addition, we thank Ayhan Aysal for his assistances in LC-MS analyses.

## **6. Conclusion**

In order to test the plant extracts, a potential drug resistant *M. tuberculosis* isolates was obtained from pulmonary tuberculosis hospital patients. The strains and isolates were then treated with plant extracts that are used for ethnopharmacological purposes. The level of the phenolic compounds and some flavonoids extracts were determined by liquid chromatography–mass spectrometry (LC-MS). The evaluation of results included the plants efficacy, their major phenolics, flavonoids and antimycobacterial activities. All plants extract showed antimycobacterial activity.

*O. acutidens, S. microstegia,* and *T. syriacus* were exhibited the lowest MIC value at 0.4 mg/mL against *M. tuberculosis* H37 Ra. *S. boissieri* and *T. syriacus* showed activity at MIC 0,4 mg/mL against *M. tuberculosis* H37 Rv. The prominent MIC and MBC values against *M. tuberculosis* H37 Rv were determined at 0,8 mg/mL for *S. boissieri* and 3,1 mg/mL for *S. cretica* subsp*. smyrnaea. S. boissieri* and *T. cilicicus* were effective against two TB-positive isolates.

The present work provides a preliminary insight into the effects of phenolics against *M. tuberculosis.* Plants of the Lamiaceae family have been shown to include new and effective constituents against *Mycobacterium tuberculosis*. Examination of these species, reported above, shows that rutin hydrate and vanillic acid were plentiful in all three extracts for these genera in Lamiaceae. All extracts of the *Origanum* species*, Salvia*, *Satureja, Stachys* and *Thymus* genera were rich in rosmarinic acid. With the exception of *S. viridis*, these species did not contain gallic acid*.* 

We suggest that phenolics and naturally occurring flavonoids (polyphenols) are mainly responsible for antimycobacterial, cytotoxicological and mutagenic activity against *M. tuberculosis*. In some plants, (*O. acutidens*, *O. sipyleum, S. microstegia,* and *Stachys byzantine)*  MIC and MBC values of CL extracts were in the same concentrations. These results might be due to several factors, such as a toxic effect caused by some compounds in the extracts. Liu et al. (2010) showed that a high concentration of cinnamic acid has toxic effects on soil bacteria. The other reason might be that the primary targets of the flavonoids have not been studied as widely in bacteria as in eukaryotes. While flavonoids affect enzyme systems such as prostaglandin, cyclooxygenase and lipoxygenase in eukaryotic cells, the bacteriocidal effect of the flavonoids might have caused the metabolic disorders on metalloenzymes by which their heavy metal atoms combine with flavonoids as ligand complexes in bacteria. These strong complexes might disrupt the metabolism of organism (Havsteen, 2002). Flavonoids are also known to have mutagenic and antitumor activities (Hodec et al., 2002; Havsteen, 2002). Quercetin affects bacteria by inhibiting the catalytic activity of DNA topoisomerase I and II (Constantinou et al., 1995; Hodec et al., 2002). Quercetin was also reported by Xu et al. (2000) and Spedding et al. (1989) to have inhibitory effects on HIV1 protease and reverse transcriptase.

It is imperative to investigate the use of new, cheaper and efficient compounds to control *Mycobacteria tuberculosis*. Recent studies have examined plants and the effectiveness of their different types of extracts on *M. tuberculosis*. Advanced research into the structure and activity relationships among naturally occurring flavonoids will yield greater understanding of their pharmacokinetics and effects on mycobacteria metabolism according to their structure. It is of great importance to determine the mechanisms of action of flavonoids on *M. tuberculosis* .

## **7. References**

326 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Lechner et al. (2008) showed that myricetin was the most efficient intensifier of INH susceptibility in all tested strains by decreasing the MIC value of INH by as much as 64-fold; the second most effective compound was quercetin. Huang et al. (1980) tested two benzenoid compounds isolated from *Ardisia japonica* in-vivo on 201 patients infected with *M. tuberculosis* (Okunade et al., 2004). They reported that both compounds showed over 80%

The authors are grateful to TUBITAK. This research was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK), TBAG (Research Grant No. 104T336). In addition, we thank Ayhan Aysal for his assistances in LC-MS analyses.

In order to test the plant extracts, a potential drug resistant *M. tuberculosis* isolates was obtained from pulmonary tuberculosis hospital patients. The strains and isolates were then treated with plant extracts that are used for ethnopharmacological purposes. The level of the phenolic compounds and some flavonoids extracts were determined by liquid chromatography–mass spectrometry (LC-MS). The evaluation of results included the plants efficacy, their major phenolics, flavonoids and antimycobacterial activities. All plants extract

*O. acutidens, S. microstegia,* and *T. syriacus* were exhibited the lowest MIC value at 0.4 mg/mL against *M. tuberculosis* H37 Ra. *S. boissieri* and *T. syriacus* showed activity at MIC 0,4 mg/mL against *M. tuberculosis* H37 Rv. The prominent MIC and MBC values against *M. tuberculosis* H37 Rv were determined at 0,8 mg/mL for *S. boissieri* and 3,1 mg/mL for *S. cretica* subsp*.* 

The present work provides a preliminary insight into the effects of phenolics against *M. tuberculosis.* Plants of the Lamiaceae family have been shown to include new and effective constituents against *Mycobacterium tuberculosis*. Examination of these species, reported above, shows that rutin hydrate and vanillic acid were plentiful in all three extracts for these genera in Lamiaceae. All extracts of the *Origanum* species*, Salvia*, *Satureja, Stachys* and *Thymus* genera were rich in rosmarinic acid. With the exception of *S. viridis*, these species

We suggest that phenolics and naturally occurring flavonoids (polyphenols) are mainly responsible for antimycobacterial, cytotoxicological and mutagenic activity against *M. tuberculosis*. In some plants, (*O. acutidens*, *O. sipyleum, S. microstegia,* and *Stachys byzantine)*  MIC and MBC values of CL extracts were in the same concentrations. These results might be due to several factors, such as a toxic effect caused by some compounds in the extracts. Liu et al. (2010) showed that a high concentration of cinnamic acid has toxic effects on soil bacteria. The other reason might be that the primary targets of the flavonoids have not been studied as widely in bacteria as in eukaryotes. While flavonoids affect enzyme systems such as prostaglandin, cyclooxygenase and lipoxygenase in eukaryotic cells, the bacteriocidal effect of the flavonoids might have caused the metabolic disorders on metalloenzymes by which their heavy metal atoms combine with flavonoids as ligand complexes in bacteria.

*smyrnaea. S. boissieri* and *T. cilicicus* were effective against two TB-positive isolates.

efficacy.

**5. Acknowledgments** 

showed antimycobacterial activity.

did not contain gallic acid*.* 

**6. Conclusion** 


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**15** 

**Cinnamic Derivatives in Tuberculosis** 

Prithwiraj De1,2, Damien Veau1,2, Florence Bedos-Belval1,2,

*(Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique),* 

Tuberculosis (TB) is a threat to worldwide public health, mainly caused by *Mycobacterium tuberculosis* (*M.tb.*) bacteria species. Despite the availability of effective treatment, tuberculosis is responsible for more than three million deaths annually worldwide. The high susceptibility of human immunodeficiency virus-infected persons to the disease (Nunn et al., 2005), the emergence of multi-drug-resistant (MDR-TB) strains (Rastogi et al., 1992, Kochi et al., 1993; Bloch et al., 1994) and extensively drug-resistant (XDR-TB) ones have brought this infectious disease into the focus of urgent scientific interest. For this reason, there is a growing need and urgency to discover new classes of chemical compounds acting with different mechanisms from those currently used. Cinnamic acid (**1; Fig. 1**) and derivatives have a century-old history as antituberculosis agents. For example, gradual improvement was observed when the TB-patients were treated with cinnamic acid (**1**) prepared from storax (Warbasse, 1894). Furthermore, in 1920s, ethylcinnamate (**2**) (Jacobson, 1919), sodium cinnamate (**3**) (Corper et al., 1920) and benzylcinnamate (**4**) (Gainsborough, 1928) were reported to be efficacious in the treatment of TB (Fig. 1). Nevertheless, we feel that this class of molecules remained underutilized until recent years. Particularly in the last two decades, there has been huge attention towards various natural and unnatural cinnamic derivatives and their antituberculosis efficacy. This chapter provides a comprehensive literature compilation concerning the synthesis so as the antituberculosis potency of various cinnamic acid, cinnamaldehyde and chalcone derivatives. We envisage that our effort in this chapter contributes a much needed and timely addition to the literature of medicinal

Fig. 1. Cinnamic acid, ethylcinnamate, sodium cinnamate and benzylcinnamate

**1. Introduction** 

research.

Stefan Chassaing1,2 and Michel Baltas1,2

*1Université de Toulouse,* 

*UPS, LSPCMIB* 

*France* 

*2CNRS, LSPCMIB,* 


## **Cinnamic Derivatives in Tuberculosis**

Prithwiraj De1,2, Damien Veau1,2, Florence Bedos-Belval1,2,

Stefan Chassaing1,2 and Michel Baltas1,2 *1Université de Toulouse, UPS, LSPCMIB (Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique), 2CNRS, LSPCMIB, France* 

#### **1. Introduction**

336 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

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*Phytochemistry*, Special Issue in memory of Professor Jeffrey B Harborne Vol.64,

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Dunder, RJ., Socca, EAR., Manzo, LP., Rozza, AL., Salvador, MJ., Pellizzon, CH., Hiruma-Lima, C.A., Luiz-Ferreira, A. & Souza-Brito, ARM. (2011). Gastroprotective and ulcer healing effects of essential oil from *Hyptis spicigera* Lam. (Lamiaceae), *Journal of Ethnopharmacology*, Vol.135, No.1, (April 2011), pp. 147-155, ISSN 0378-

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(2010). Acaricidal properties of the essential oil from *Hesperozygis ringens* (Lamiaceae) on the cattle tick *Riphicephalus* (Boophilus) *microplus*, *Bioresource*

antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs, *Food Chemistry*, Vol.83, No.2, (November 2003), pp. 255-262, ISSN 0308-8146.

Tuberculosis (TB) is a threat to worldwide public health, mainly caused by *Mycobacterium tuberculosis* (*M.tb.*) bacteria species. Despite the availability of effective treatment, tuberculosis is responsible for more than three million deaths annually worldwide. The high susceptibility of human immunodeficiency virus-infected persons to the disease (Nunn et al., 2005), the emergence of multi-drug-resistant (MDR-TB) strains (Rastogi et al., 1992, Kochi et al., 1993; Bloch et al., 1994) and extensively drug-resistant (XDR-TB) ones have brought this infectious disease into the focus of urgent scientific interest. For this reason, there is a growing need and urgency to discover new classes of chemical compounds acting with different mechanisms from those currently used. Cinnamic acid (**1; Fig. 1**) and derivatives have a century-old history as antituberculosis agents. For example, gradual improvement was observed when the TB-patients were treated with cinnamic acid (**1**) prepared from storax (Warbasse, 1894). Furthermore, in 1920s, ethylcinnamate (**2**) (Jacobson, 1919), sodium cinnamate (**3**) (Corper et al., 1920) and benzylcinnamate (**4**) (Gainsborough, 1928) were reported to be efficacious in the treatment of TB (Fig. 1). Nevertheless, we feel that this class of molecules remained underutilized until recent years. Particularly in the last two decades, there has been huge attention towards various natural and unnatural cinnamic derivatives and their antituberculosis efficacy. This chapter provides a comprehensive literature compilation concerning the synthesis so as the antituberculosis potency of various cinnamic acid, cinnamaldehyde and chalcone derivatives. We envisage that our effort in this chapter contributes a much needed and timely addition to the literature of medicinal research.

Fig. 1. Cinnamic acid, ethylcinnamate, sodium cinnamate and benzylcinnamate

Cinnamic Derivatives in Tuberculosis 339

proved that **1** is not bactericidal. The dose-dependent effect of cerulenin (**16**) and *trans*cinnamic acid on *M.tb.* viability showed (Rastogi *et al.* 1996) that cinnamic acid was not bactericidal even at concentrations as high as 200 µg/mL, whereas cerulenin was

Thus, the sub minimum inhibitory concentrations (sub-MIC) of both **16** and **1** used in synergy experiments (fixed concentrations of only 1 µg/mL) were not due to any direct effect of these two inhibitors on *tubercle bacilli* (TB). Out of the various drug combinations screened, those with **1** gave the best results. For example, enhancement of drug activity was even observed with the drug-resistant strain 92-0492 (resistant to isoniazid (**11**) & rifampin (**12**)) when **1** at sub-MIC concentrations was used in combination with antibiotics such as ofloxacine (**13**), clofazimine (**14**) and amikacine (**15**). Cerulenin (**16**) is a known antifungal antibiotic that inhibits fatty acid and steroid biosynthesis. In fatty acid synthesis, **1** proved to bind in equimolar ratio to β-keto-acyl-ACP synthase, one of the seven moieties of fatty acid synthase, blocking the interaction of malonyl-CoA (Nomura et al*.,* 1972; Omura, 1976). It is therefore likely that the inhibitory effects that were observed in the synergy study resulted from the inhibition of fatty acid synthesis. However, the mode of action for **1** is still unknown. In a previous study with *M. avium* (Rastogi et al*.,*  1994), it was suspected that **1** might have inhibitory effects because of its structural similarity to phenylalanine. Because of that similarity, **1** would inhibit glycopeptidelipid (GPL) biosynthesis, therefore increasing cell wall permeability and enhancing the inhibitory effect of antimycobacterial drugs. As *M.tb.* does not synthesize GPL antigens, this reasoning does not fully fit with this bacterial species*.* Apparently other sites are also

bactericidal only at concentrations above 50 µg/mL.

Fig. 3. Known antituberculosis drugs

Fig. 4. Known antibiotics used in synergy studies

#### **2. Cinnamic acid derivatives as anti-TB agents**

*trans*-Cinnamic acid (1) has a long history of human use as a component of plant-derived scents and flavourings (Hoskins, 1984). It belongs to the class of auxin, which is recognized as plant hormones regulating cell growth and differentiation (Thimann, 1969). The cinnamoyl functionality is also present in a variety of secondary metabolites of phenylpropanoid biosynthetic origin. Those containing a sesquiterpenyl, monoterpenyl and isopentenyl chain attached to a 4-hydroxy group represent quite a rare group of natural products (Epifano et al., 2007).

Fig. 2. Cinnamic acid and its natural phenolic-analogues

The hydroxyl cinnamic acids such as *p*-coumaric acid (**5**), caffeic acid (**6**), ferulic acid (**7**), sinapic acid (**8**) (**Fig. 2**) are natural products arising from the deamination of the phenyl alanine (**9**) (**Scheme 1**) (Kroon & Williamson, 1999). Besides, they are important constituents in the biochemical pathway in plants leading to the lignin (Humphreys & Chapple, 2002; Boerjan et al*.*, 2003), the second most abundant biopolymer after cellulose (Whetten et al., 1998) resulting mainly from the oxidative polymerization (Freudenberg, 1959) of the three hydroxycinnamyl alcohols, namely coumaryl (**10a**), coniferyl (**10b**) and sinapyl alcohols (**10c**). These key cinnamyl alcohols are produced through two successive enzyme-catalyzed reductions starting from the corresponding cinnamyl SCoA-esters. In recent years, *trans*cinnamic acid derivatives have also attracted much attention due to their antioxidative (Chung & Shin, 2007), antitumor (De et al., 2011a) and antimicrobial (Naz et al., 2006; Carvalho et al., 2008) properties.

Scheme 1. Lignin biosynthesis pathway

#### **2.1 Cinnamic acid**

In an attempt to develop a new strategy to circumvent MDR-TB by augmenting the potential of the existing drugs (Rastogi et al. 1994), *trans*-cinnamic acid (**1**) was used along with known antituberculous drugs such as isoniazid (**11**), rifampin (**12**), ofloxacine (**13**) or clofazimine (**14**) (**Fig**. **3**). Interestingly, a synergistic increase was observed in the activity of various drugs against *Mycobacterium avium*. The synergistic activity of **1** (Rastogi *et al.* 1994) with a variety of drugs was manifested even with drug resistant isolates. Importantly, it was

*trans*-Cinnamic acid (1) has a long history of human use as a component of plant-derived scents and flavourings (Hoskins, 1984). It belongs to the class of auxin, which is recognized as plant hormones regulating cell growth and differentiation (Thimann, 1969). The cinnamoyl functionality is also present in a variety of secondary metabolites of phenylpropanoid biosynthetic origin. Those containing a sesquiterpenyl, monoterpenyl and isopentenyl chain attached to a 4-hydroxy group represent quite a rare group of natural

The hydroxyl cinnamic acids such as *p*-coumaric acid (**5**), caffeic acid (**6**), ferulic acid (**7**), sinapic acid (**8**) (**Fig. 2**) are natural products arising from the deamination of the phenyl alanine (**9**) (**Scheme 1**) (Kroon & Williamson, 1999). Besides, they are important constituents in the biochemical pathway in plants leading to the lignin (Humphreys & Chapple, 2002; Boerjan et al*.*, 2003), the second most abundant biopolymer after cellulose (Whetten et al., 1998) resulting mainly from the oxidative polymerization (Freudenberg, 1959) of the three hydroxycinnamyl alcohols, namely coumaryl (**10a**), coniferyl (**10b**) and sinapyl alcohols (**10c**). These key cinnamyl alcohols are produced through two successive enzyme-catalyzed reductions starting from the corresponding cinnamyl SCoA-esters. In recent years, *trans*cinnamic acid derivatives have also attracted much attention due to their antioxidative (Chung & Shin, 2007), antitumor (De et al., 2011a) and antimicrobial (Naz et al., 2006;

In an attempt to develop a new strategy to circumvent MDR-TB by augmenting the potential of the existing drugs (Rastogi et al. 1994), *trans*-cinnamic acid (**1**) was used along with known antituberculous drugs such as isoniazid (**11**), rifampin (**12**), ofloxacine (**13**) or clofazimine (**14**) (**Fig**. **3**). Interestingly, a synergistic increase was observed in the activity of various drugs against *Mycobacterium avium*. The synergistic activity of **1** (Rastogi *et al.* 1994) with a variety of drugs was manifested even with drug resistant isolates. Importantly, it was

**2. Cinnamic acid derivatives as anti-TB agents** 

Fig. 2. Cinnamic acid and its natural phenolic-analogues

products (Epifano et al., 2007).

Carvalho et al., 2008) properties.

Scheme 1. Lignin biosynthesis pathway

**2.1 Cinnamic acid** 

proved that **1** is not bactericidal. The dose-dependent effect of cerulenin (**16**) and *trans*cinnamic acid on *M.tb.* viability showed (Rastogi *et al.* 1996) that cinnamic acid was not bactericidal even at concentrations as high as 200 µg/mL, whereas cerulenin was bactericidal only at concentrations above 50 µg/mL.

Fig. 4. Known antibiotics used in synergy studies

Thus, the sub minimum inhibitory concentrations (sub-MIC) of both **16** and **1** used in synergy experiments (fixed concentrations of only 1 µg/mL) were not due to any direct effect of these two inhibitors on *tubercle bacilli* (TB). Out of the various drug combinations screened, those with **1** gave the best results. For example, enhancement of drug activity was even observed with the drug-resistant strain 92-0492 (resistant to isoniazid (**11**) & rifampin (**12**)) when **1** at sub-MIC concentrations was used in combination with antibiotics such as ofloxacine (**13**), clofazimine (**14**) and amikacine (**15**). Cerulenin (**16**) is a known antifungal antibiotic that inhibits fatty acid and steroid biosynthesis. In fatty acid synthesis, **1** proved to bind in equimolar ratio to β-keto-acyl-ACP synthase, one of the seven moieties of fatty acid synthase, blocking the interaction of malonyl-CoA (Nomura et al*.,* 1972; Omura, 1976). It is therefore likely that the inhibitory effects that were observed in the synergy study resulted from the inhibition of fatty acid synthesis. However, the mode of action for **1** is still unknown. In a previous study with *M. avium* (Rastogi et al*.,*  1994), it was suspected that **1** might have inhibitory effects because of its structural similarity to phenylalanine. Because of that similarity, **1** would inhibit glycopeptidelipid (GPL) biosynthesis, therefore increasing cell wall permeability and enhancing the inhibitory effect of antimycobacterial drugs. As *M.tb.* does not synthesize GPL antigens, this reasoning does not fully fit with this bacterial species*.* Apparently other sites are also

Cinnamic Derivatives in Tuberculosis 341

It is well known that triterpenes exhibit moderate to high *in vitro* antimycobacterial activity against *M. tb*. (Copp & Pearce, 2007; Okunade, Elvin-Lewis & Lewis, 2004). The modification of natural triterpenes such as betulinic acid (**21**), oleanolic acid (**22**) and ursolic acid (**23**) through introduction of cinnamoyl frames at the C-3 position has been reported (**Scheme 2**) (Tanachatchairatana et al., 2008). Different cinnamoyl derivatives such as cinnamate, *p*coumarate, ferulate, caffeate and *p*-chlorocinnamate esters of the above mentioned triterpenes were synthesized by reacting with the suitable cinnamoyl chlorides in the presence of 4-*N*,*N*-dimethylaminopyridine (DMAP) in benzene. All the hydroxyl-cinnamic acids were acetylated to protect the phenolic group before generating the corresponding acid chlorides followed by coupling with the triterpenes. The hydroxycinnamate derivatives of the triterpenes (**21d,f,h**; **22d,f,h**; **23d,f,h**) were easily obtained by deacetylation of the acetylated derivatives (**21c,e,g**; **22c,e,g**; **23c,e,g**) using K2CO3 in methanol. The biological results indicated that the introduction of unsubstituted or *p*-chlorinated cinnamate ester functionality (**21a,b**; **22a,b**; **23a,b**) led to inactive compounds (MIC>200 µg/mL) or without any improvement in the antimycobacterial activity of the native triterpenes. Interestingly, the results also indicated that introduction of the *p*-coumarate moiety at the C-3 position of the triterpenes (**21d**, **22d**, **23d**) resulted in an 8-fold increase in antimycobacterial activity of the parent triterpenes **21** (MIC = 50 µg/mL) and **22** (MIC = 50 µg/mL), and a 2-fold increase in the activity of the triterpene **23** (MIC = 12.5 µg/mL). Introduction of a ferulate moiety (**21h**, **22h**, **23h**) resulted in a 4-fold increase in activity only in case of **23**. However, the presence of a *p*-hydroxy group plays a crucial role on the high antimycobacterial activity because its methylation and acetylation proved to decrease antimycobacterial activity in a

significant manner, caffeate esters **21e,g** and **22e,g** being the exceptions.

Scheme 2. Cinnamate-based triterpenes and their biological activities

Rifampin (RIF; **12; Fig. 3**) is one of the most important drugs in TB treatment. In search for new compounds with structural modifications of existing lead drugs, the presence of a cinnamoyl moiety on rifampin's piperazinyl framework instead of a methyl group,

**2.3.2 Cinnamic amide derivatives** 

**2.3 Synthetic compounds** 

**2.3.1 Cinnamic ester derivatives** 

being affected by **1**, which in turn enhance the susceptibility of the organism to the effects of the antimycobacterial drugs. Although, *trans*-cinnamic acid (**1**) was used to treat tuberculosis before antimycobacterial chemotherapy was used (Ryan, 1992), this was the first example of MDR-TB activity in synergy with other drugs but the mechanism of action still remains unknown.

#### **2.2 Natural compounds**

In another context, a cinnamoyl ester was identified to be important in glycoside extracts of a native North American prairie plant named *Ipomoea leptophylla*. In fact, the organic soluble extracts from its leaves showed (Barnes et al*.* 2003) *in vitro* activity against *M.tb*. Through a bioassay-guided fractionation of these extracts, the authors isolated leptophyllin A (**18**), a resin glycoside bearing a *trans*-cinnamic residue attached to one rhamnose moiety (**Fig. 5**). This compound showed 13% inhibition at 6.25 *µ*g/mL against *M. tb.* in the *in vitro* anti-TB assay. Furthermore, the bioassay results indicated that the cinnamic acid residue is required for the observed antimicrobial activity as an analogous compound bearing no cinnamic acid residue, leptophyllin B (**19**), also isolated from the same source, showed no *in vitro* activity. Recently, some bioactive styryllactones and alkaloids were isolated from flowers of *Goniothalamus laoticus* (Lekphrom et al. 2009). In particular, the authors isolated a styryllactone derivative, namely howiinin A (**20**), by fractionation of the ethyl acetate and methanol extracts from the flowers of this species (**Fig**. **6**). While inactive against *Plasmodium falciparum*, this compound possessing a cinnamoyl ester moiety showed an interesting anti-TB activity (MIC = 6.25 µg/mL) when tested against *M.tb.* strain H37Ra.

Fig. 5. Resin glycosides from *I. Leptophylla* 

Fig. 6. Structure of Howiinin A

#### **2.3 Synthetic compounds**

340 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

being affected by **1**, which in turn enhance the susceptibility of the organism to the effects of the antimycobacterial drugs. Although, *trans*-cinnamic acid (**1**) was used to treat tuberculosis before antimycobacterial chemotherapy was used (Ryan, 1992), this was the first example of MDR-TB activity in synergy with other drugs but the mechanism of

In another context, a cinnamoyl ester was identified to be important in glycoside extracts of a native North American prairie plant named *Ipomoea leptophylla*. In fact, the organic soluble extracts from its leaves showed (Barnes et al*.* 2003) *in vitro* activity against *M.tb*. Through a bioassay-guided fractionation of these extracts, the authors isolated leptophyllin A (**18**), a resin glycoside bearing a *trans*-cinnamic residue attached to one rhamnose moiety (**Fig. 5**). This compound showed 13% inhibition at 6.25 *µ*g/mL against *M. tb.* in the *in vitro* anti-TB assay. Furthermore, the bioassay results indicated that the cinnamic acid residue is required for the observed antimicrobial activity as an analogous compound bearing no cinnamic acid residue, leptophyllin B (**19**), also isolated from the same source, showed no *in vitro* activity. Recently, some bioactive styryllactones and alkaloids were isolated from flowers of *Goniothalamus laoticus* (Lekphrom et al. 2009). In particular, the authors isolated a styryllactone derivative, namely howiinin A (**20**), by fractionation of the ethyl acetate and methanol extracts from the flowers of this species (**Fig**. **6**). While inactive against *Plasmodium falciparum*, this compound possessing a cinnamoyl ester moiety showed an interesting anti-

TB activity (MIC = 6.25 µg/mL) when tested against *M.tb.* strain H37Ra.

Fig. 5. Resin glycosides from *I. Leptophylla* 

Fig. 6. Structure of Howiinin A

action still remains unknown.

**2.2 Natural compounds** 

#### **2.3.1 Cinnamic ester derivatives**

It is well known that triterpenes exhibit moderate to high *in vitro* antimycobacterial activity against *M. tb*. (Copp & Pearce, 2007; Okunade, Elvin-Lewis & Lewis, 2004). The modification of natural triterpenes such as betulinic acid (**21**), oleanolic acid (**22**) and ursolic acid (**23**) through introduction of cinnamoyl frames at the C-3 position has been reported (**Scheme 2**) (Tanachatchairatana et al., 2008). Different cinnamoyl derivatives such as cinnamate, *p*coumarate, ferulate, caffeate and *p*-chlorocinnamate esters of the above mentioned triterpenes were synthesized by reacting with the suitable cinnamoyl chlorides in the presence of 4-*N*,*N*-dimethylaminopyridine (DMAP) in benzene. All the hydroxyl-cinnamic acids were acetylated to protect the phenolic group before generating the corresponding acid chlorides followed by coupling with the triterpenes. The hydroxycinnamate derivatives of the triterpenes (**21d,f,h**; **22d,f,h**; **23d,f,h**) were easily obtained by deacetylation of the acetylated derivatives (**21c,e,g**; **22c,e,g**; **23c,e,g**) using K2CO3 in methanol. The biological results indicated that the introduction of unsubstituted or *p*-chlorinated cinnamate ester functionality (**21a,b**; **22a,b**; **23a,b**) led to inactive compounds (MIC>200 µg/mL) or without any improvement in the antimycobacterial activity of the native triterpenes. Interestingly, the results also indicated that introduction of the *p*-coumarate moiety at the C-3 position of the triterpenes (**21d**, **22d**, **23d**) resulted in an 8-fold increase in antimycobacterial activity of the parent triterpenes **21** (MIC = 50 µg/mL) and **22** (MIC = 50 µg/mL), and a 2-fold increase in the activity of the triterpene **23** (MIC = 12.5 µg/mL). Introduction of a ferulate moiety (**21h**, **22h**, **23h**) resulted in a 4-fold increase in activity only in case of **23**. However, the presence of a *p*-hydroxy group plays a crucial role on the high antimycobacterial activity because its methylation and acetylation proved to decrease antimycobacterial activity in a significant manner, caffeate esters **21e,g** and **22e,g** being the exceptions.

Scheme 2. Cinnamate-based triterpenes and their biological activities

#### **2.3.2 Cinnamic amide derivatives**

Rifampin (RIF; **12; Fig. 3**) is one of the most important drugs in TB treatment. In search for new compounds with structural modifications of existing lead drugs, the presence of a cinnamoyl moiety on rifampin's piperazinyl framework instead of a methyl group,

Cinnamic Derivatives in Tuberculosis 343

(REMA) against *M. tb.* H37Rv (MIC = 6.5 µM) along with good safety profile (CC50 = 340 µM) in VERO cell line. Importantly, analysis of structure–activity relationships revealed that both steric and electronic parameters play major role in the activity of this series of

PhOH, SOCl2

NH NH2 NH

> H N

NH NH NH O

**28**; MIC = 6.5 µM

OPh O

**27a**

OMe

Et3N, EtOH, MW, 60W

H N

OMe

OH O

MeO MeO

OMe

MeO

MeO

A series of 1-[(2,3-dichloroanilinomalonyl)-3-(*N*-2'-cyanoethyl)-2-(*N*-cinnamoyl) 2,3-Dichloroanilino)]-5-phenyl pyrazolines (**38a-e**) have been synthesized from *N*-cinnamoyl-*N*-2'-cyanoethyl-2,3-dichloroaniline (**37**) in the presence of 2-[(*N*-cinnamoyl) 2,3 dichloroanilido] acetohydrazides (**33a-e**) and acetic acid in dioxane (**Scheme 4**) (Sharma et al., 2010, 2011). These compounds have been tested for *in vitro* antitubercular activity on

The compounds (**38a-e**) inhibited the growth of *M.tb.* at 100 µg/mL concentration. The acylhydrazide (**33a**) was also coupled with some aromatic aldehydes (**29a-c**) in the presence of catalytic H2SO4 in ethanol. The resulting hydrazones (**34a-c**) were also found to have anti-

Using a molecular hybridization approach, a series of cinnamide derivatives (**39a**-**42a**) was designed as potential antimycobacterial agents (Kakwani et al*.* 2011). The diamine moiety of ethambutol (**17; Fig. 4**) and its other analogs proved to be a key feature. Various diamines (**39-42**) were coupled with **1** using ethylchloroformate and triethylamine to obtain cinnamide derivatives (**39a**-**42a**) (**Scheme 5**). The MICs of all synthesized compounds were determined against *M.tb.* H37Rv using Resazurin Microtitre plate Assay (REMA) method. The synthesized molecules (**39a**-**42a**) showed good to moderate activities with MIC in the range of 5–150 μM and good safety profiles. The most potent compound **39a**, having MIC of 5.1 μM (cytotoxicity measured on VERO cells line C1008 (CC50) = 618 μM) exhibited synergy with rifampin (**12**). Under similar conditions ethambutol (**17**) showed MIC of 15.3 μM with a

**27**

H EtOH, MW, 60W

**25 25a**

Scheme 3. Synthetic route for the synthesis of phenylacrylamide derivatives

O

OMe

+

H2N

NH NH2 NH .HCl

**26**

compounds.

MeO

*M.tb***.** H37Rv strains.

CC50 value of 1470 μM.

TB activity.

furnishing 3-(4-cinnamylpiperazinyl-iminomethyl)rifamycin derivative (**24**; rifamycin SV (T9); **Fig. 7**), resulted in enhanced antimycobacterial activity (Reddy *et al*. 1995; Velichka et al. 2010). The antimycobacterial activities of **24** on 20 susceptible and MDR-strains of *M.tb.*  and 20 *M. avium* complex (MAC) strains were investigated (Dimova *et al.* 2010). The radiometric MICs of T9 for *M.tb.* were significantly lower than those of RIF. The MICs of T9 and RIF at which 90% of the RIF-susceptible strains were inhibited were <0.25 and <0.5 µg/mL, respectively. Compound **24** had better activity against MAC strains, and the MIC at which 90% of the MAC strains were inhibited was <0.125 µg/mL, while that of RIF was <2.0 µg/mL. Compound **24** also showed high *in vitro* bactericidal and intracellular activities which were significantly superior to those of RIF against both *M.tb.* and MAC strains. More importantly, **24** showed excellent *in vivo* activity against *M.tb.* H37Rv compared to RIF in both the lungs and spleens of C57BL/6 mice, indicating the potential therapeutic value of **24** in the treatment of mycobacterial infections.

#### Fig. 7. Structure of Rifamycin SV(T9)

In an attempt to find novel compounds active against TB, a series of phenylacrylamides designed by molecular hybridization of *trans*-cinnamic acids and guanylhydrazones were synthesized and antiTB efficacy were evaluated (**Scheme 3**) (Bairwa et al., 2010). While cinnamic acids are already known for their antituberculosis efficacy, guanylhydrazones have been shown to have antimicrobial activity including an interesting gram-negative bacterial endotoxin lipopolysaccharide (LPS) sequestering activity (Gadad et al. 2000; Wu et al. 2009). *M. tb.* contains lipoarabinomannan (LAM), a complex lipid glycoprotein anchored to the cell membrane by phosphatidylinositol which has structural and functional similarity to LPS, including the presence of anionic phosphate groups (Zhang et al., 1994). Biosynthesis of LAM is known to be a target for several antituberculosis agents, including the first line antitubercular agent, ethambutol (**17**; **Fig. 4**) (Scherman et al. 1995; Heijenoort, 2001). For the synthesis of the most active phenylacrylamide derivative (**28**; **Scheme 3**), the required guanylhydrazone (**25a**) was prepared by the microwave-assisted reaction of 3,4-dimethoxy benzaldehyde (**25**) with guanylhydrazine hydrochloride (**26**). In parallel, the phenyl 4-methoxycinnamate (**27a**) was prepared by esterification of 4-methoxycinnamic acid (**27**) via its treatment by phenol and thionyl chloride. Finally, the coupling of equimolar quantities of guanylhydrazone (**25a**) with phenylcinnamate (**27a**), was performed under microwave irradiation in the presence of triethylamine and ethanol as solvent to afford the target derivative (*E*)-*N*-(((*E*)-2-(3,4 dimethoxybenzylidene)hydrazinyl)(imino)methyl)-3-(4-methoxyphenyl)acrylamide (**28**). Compound **28** was found to be active when tested on resazurin microtiter plate assay

furnishing 3-(4-cinnamylpiperazinyl-iminomethyl)rifamycin derivative (**24**; rifamycin SV (T9); **Fig. 7**), resulted in enhanced antimycobacterial activity (Reddy *et al*. 1995; Velichka et al. 2010). The antimycobacterial activities of **24** on 20 susceptible and MDR-strains of *M.tb.*  and 20 *M. avium* complex (MAC) strains were investigated (Dimova *et al.* 2010). The radiometric MICs of T9 for *M.tb.* were significantly lower than those of RIF. The MICs of T9 and RIF at which 90% of the RIF-susceptible strains were inhibited were <0.25 and <0.5 µg/mL, respectively. Compound **24** had better activity against MAC strains, and the MIC at which 90% of the MAC strains were inhibited was <0.125 µg/mL, while that of RIF was <2.0 µg/mL. Compound **24** also showed high *in vitro* bactericidal and intracellular activities which were significantly superior to those of RIF against both *M.tb.* and MAC strains. More importantly, **24** showed excellent *in vivo* activity against *M.tb.* H37Rv compared to RIF in both the lungs and spleens of C57BL/6 mice, indicating the potential therapeutic value of **24**

In an attempt to find novel compounds active against TB, a series of phenylacrylamides designed by molecular hybridization of *trans*-cinnamic acids and guanylhydrazones were synthesized and antiTB efficacy were evaluated (**Scheme 3**) (Bairwa et al., 2010). While cinnamic acids are already known for their antituberculosis efficacy, guanylhydrazones have been shown to have antimicrobial activity including an interesting gram-negative bacterial endotoxin lipopolysaccharide (LPS) sequestering activity (Gadad et al. 2000; Wu et al. 2009). *M. tb.* contains lipoarabinomannan (LAM), a complex lipid glycoprotein anchored to the cell membrane by phosphatidylinositol which has structural and functional similarity to LPS, including the presence of anionic phosphate groups (Zhang et al., 1994). Biosynthesis of LAM is known to be a target for several antituberculosis agents, including the first line antitubercular agent, ethambutol (**17**; **Fig. 4**) (Scherman et al. 1995; Heijenoort, 2001). For the synthesis of the most active phenylacrylamide derivative (**28**; **Scheme 3**), the required guanylhydrazone (**25a**) was prepared by the microwave-assisted reaction of 3,4-dimethoxy benzaldehyde (**25**) with guanylhydrazine hydrochloride (**26**). In parallel, the phenyl 4-methoxycinnamate (**27a**) was prepared by esterification of 4-methoxycinnamic acid (**27**) via its treatment by phenol and thionyl chloride. Finally, the coupling of equimolar quantities of guanylhydrazone (**25a**) with phenylcinnamate (**27a**), was performed under microwave irradiation in the presence of triethylamine and ethanol as solvent to afford the target derivative (*E*)-*N*-(((*E*)-2-(3,4 dimethoxybenzylidene)hydrazinyl)(imino)methyl)-3-(4-methoxyphenyl)acrylamide (**28**). Compound **28** was found to be active when tested on resazurin microtiter plate assay

in the treatment of mycobacterial infections.

Fig. 7. Structure of Rifamycin SV(T9)

(REMA) against *M. tb.* H37Rv (MIC = 6.5 µM) along with good safety profile (CC50 = 340 µM) in VERO cell line. Importantly, analysis of structure–activity relationships revealed that both steric and electronic parameters play major role in the activity of this series of compounds.

Scheme 3. Synthetic route for the synthesis of phenylacrylamide derivatives

A series of 1-[(2,3-dichloroanilinomalonyl)-3-(*N*-2'-cyanoethyl)-2-(*N*-cinnamoyl) 2,3-Dichloroanilino)]-5-phenyl pyrazolines (**38a-e**) have been synthesized from *N*-cinnamoyl-*N*-2'-cyanoethyl-2,3-dichloroaniline (**37**) in the presence of 2-[(*N*-cinnamoyl) 2,3 dichloroanilido] acetohydrazides (**33a-e**) and acetic acid in dioxane (**Scheme 4**) (Sharma et al., 2010, 2011). These compounds have been tested for *in vitro* antitubercular activity on *M.tb***.** H37Rv strains.

The compounds (**38a-e**) inhibited the growth of *M.tb.* at 100 µg/mL concentration. The acylhydrazide (**33a**) was also coupled with some aromatic aldehydes (**29a-c**) in the presence of catalytic H2SO4 in ethanol. The resulting hydrazones (**34a-c**) were also found to have anti-TB activity.

Using a molecular hybridization approach, a series of cinnamide derivatives (**39a**-**42a**) was designed as potential antimycobacterial agents (Kakwani et al*.* 2011). The diamine moiety of ethambutol (**17; Fig. 4**) and its other analogs proved to be a key feature. Various diamines (**39-42**) were coupled with **1** using ethylchloroformate and triethylamine to obtain cinnamide derivatives (**39a**-**42a**) (**Scheme 5**). The MICs of all synthesized compounds were determined against *M.tb.* H37Rv using Resazurin Microtitre plate Assay (REMA) method. The synthesized molecules (**39a**-**42a**) showed good to moderate activities with MIC in the range of 5–150 μM and good safety profiles. The most potent compound **39a**, having MIC of 5.1 μM (cytotoxicity measured on VERO cells line C1008 (CC50) = 618 μM) exhibited synergy with rifampin (**12**). Under similar conditions ethambutol (**17**) showed MIC of 15.3 μM with a CC50 value of 1470 μM.

Cinnamic Derivatives in Tuberculosis 345

oxadiazoles **45a-e**. Compounds **45a-d** were found to be more active against *M.tb.* H37Rv than

Benzimidazole scaffold being an important pharmacophore and privileged structure in medicinal chemistry (Khalafi-Nezhad et al*.*, 2005; Evans et al. 1988), a new series of 5- (nitro/bromo)-styryl-2-benzimidazoles (**49a-f, 50a-f**; **Scheme 7**) was synthesized (Shingalapur et al., 2009) by simple condensation of 5-(nitro/bromo)-*O*-phenylenediamine (**46, 47**) with *trans*-cinnamic acids (**48a-f**) in ethylene glycol for 6 h at around 200°C (**Scheme 7**). The *in vitro* anti-TB activities of compounds **49a-f** and **50a-f** on the *M.tb.* H37Rv were determined at 7.25 µg/mL concentration. Interestingly, the bromo-substituted

The sequestration of iron is a part of the non-specific mammalian immune response and thus, metal uptake and regulation of metal-ion concentrations are the key features of hostpathogen interactions (Agranoff & Krishna, 2004). Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses (Neilands, 1995; Cornelis & Andrews, 2010). Siderophores are considered amongst the strongest soluble Fe(III)-binding agents known. Siderophores, produced by mycobacteria itself, were used (Guo et al., 2002) to target iron transport processes essential for the growth and survival of *M. tb.* Targeting the iron transport processes of *M.tb.* is challenging for several reasons. The complexity of the mycobactin architecture itself poses a daunting synthetic challenge, which hampers the generation of conjugates (Xu & Miller, 1998). Further, the iron transport mechanism involves an "iron-handoff" between two siderophore families, the exochelins and the mycobactins. In low iron environments, *M.tb.*  biosynthesizes and secretes hydrophilic exochelins (e.g., Mycobactin J (**51**); **Scheme 8**) to bind exogenous ferric ion. The iron-complex is then transferred to intracellular siderophores (*i.e*. the mycobactins) which are lipophilic chelators associated with the

benzimidazole derivatives (**50a-f**) exhibited the best results with 63-83% inhibition.

Scheme 6. Synthesis of various 1,3,4-oxadiazoles and 5-oxo-imidazolines

the cinnamic derivative **45e** at 12.5 µg/mL.

**2.3.4 Cinnamic benzimidazole derivatives** 

Scheme 7. Synthesis of styryl-2-benzimidazoles series

**2.3.5 Cinnamic acid hydroxamic derivatives** 

Scheme 4. Synthesis of cinnamoyl pyrazolines and analogs

Scheme 5. Synthesis of cinnamides from diamines

#### **2.3.3 Cinnamic oxadiazole derivatives**

The synthesis of some 1,3,4-oxadiazoles and oxo-imidazolines compounds as potent biologically active agents has been reported (Joshi et al., 1997). The synthetic routes are presented in **Scheme 6**. The common precursor **44** was obtained through condensation of 5 nitro-*o*-benzoylene-2,1-benzimidazole (**43**) with hydrazine hydrate. The cyclocondensation reaction of different aromatic acids with **44** in the presence of POCl3 afforded 1,3,4-

Scheme 4. Synthesis of cinnamoyl pyrazolines and analogs

Scheme 5. Synthesis of cinnamides from diamines

The synthesis of some 1,3,4-oxadiazoles and oxo-imidazolines compounds as potent biologically active agents has been reported (Joshi et al., 1997). The synthetic routes are presented in **Scheme 6**. The common precursor **44** was obtained through condensation of 5 nitro-*o*-benzoylene-2,1-benzimidazole (**43**) with hydrazine hydrate. The cyclocondensation reaction of different aromatic acids with **44** in the presence of POCl3 afforded 1,3,4-

**2.3.3 Cinnamic oxadiazole derivatives** 

oxadiazoles **45a-e**. Compounds **45a-d** were found to be more active against *M.tb.* H37Rv than the cinnamic derivative **45e** at 12.5 µg/mL.

Scheme 6. Synthesis of various 1,3,4-oxadiazoles and 5-oxo-imidazolines

#### **2.3.4 Cinnamic benzimidazole derivatives**

Benzimidazole scaffold being an important pharmacophore and privileged structure in medicinal chemistry (Khalafi-Nezhad et al*.*, 2005; Evans et al. 1988), a new series of 5- (nitro/bromo)-styryl-2-benzimidazoles (**49a-f, 50a-f**; **Scheme 7**) was synthesized (Shingalapur et al., 2009) by simple condensation of 5-(nitro/bromo)-*O*-phenylenediamine (**46, 47**) with *trans*-cinnamic acids (**48a-f**) in ethylene glycol for 6 h at around 200°C (**Scheme 7**). The *in vitro* anti-TB activities of compounds **49a-f** and **50a-f** on the *M.tb.* H37Rv were determined at 7.25 µg/mL concentration. Interestingly, the bromo-substituted benzimidazole derivatives (**50a-f**) exhibited the best results with 63-83% inhibition.

Scheme 7. Synthesis of styryl-2-benzimidazoles series

#### **2.3.5 Cinnamic acid hydroxamic derivatives**

The sequestration of iron is a part of the non-specific mammalian immune response and thus, metal uptake and regulation of metal-ion concentrations are the key features of hostpathogen interactions (Agranoff & Krishna, 2004). Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses (Neilands, 1995; Cornelis & Andrews, 2010). Siderophores are considered amongst the strongest soluble Fe(III)-binding agents known. Siderophores, produced by mycobacteria itself, were used (Guo et al., 2002) to target iron transport processes essential for the growth and survival of *M. tb.* Targeting the iron transport processes of *M.tb.* is challenging for several reasons. The complexity of the mycobactin architecture itself poses a daunting synthetic challenge, which hampers the generation of conjugates (Xu & Miller, 1998). Further, the iron transport mechanism involves an "iron-handoff" between two siderophore families, the exochelins and the mycobactins. In low iron environments, *M.tb.*  biosynthesizes and secretes hydrophilic exochelins (e.g., Mycobactin J (**51**); **Scheme 8**) to bind exogenous ferric ion. The iron-complex is then transferred to intracellular siderophores (*i.e*. the mycobactins) which are lipophilic chelators associated with the

Cinnamic Derivatives in Tuberculosis 347

*trans*-Cinnamic acid hydrazide derivatives were presented as potential antituberculosis agents (Carvalho et al. 2008). The authors designed and explored the introduction of the *trans*-cinnamic moiety into isoniazid (**11**) core structure to ameliorate its activity. Isosteric substitution of the pyridine ring of **11** was also investigated by these authors. The synthetic route (**Scheme 9**) used for the preparation of the target compounds is rapid and relies on the formation/use of *p*-nitrophenyl esters (**58a-d**) as activated forms of cinnamic acid derivatives (**Scheme 9**). These stable intermediates (**58a-d**) were prepared by treating the appropriate cinnamic acid (**57a-d**) with thionyl chloride in the presence of 4-nitro-phenol. The target hydrazides (**11a-d**, **59a-d**; **Scheme 9**) were then obtained in good yields by coupling the so-formed activated acids (**58a-d**) with either acylhydrazide **11** or **59**. The anti-TB activities of these compounds were assessed against *M.tb*. Almost all of the isonicotinic derivatives **11a-d** were sensitive in the minimum concentration tested (MIC = 3.12 µg/mL). Nevertheless, all benzoic acid derivatives **59a-d** were much less active, thus reinforcing the pharmacophoric contribution of the isonicotinic moiety. Importantly, the authors identified

**2.3.6 Cinnamic acid hydrazide, thioester and other derivatives** 

that the 4-methoxycinnamic derivatives promote the better activity.

Scheme 9. Synthetic route for the preparation of the cinnamoyl hydrazides

In our recent effort, we have synthesized some 4-alkoxycinnamic acid thioesters, amides, hydrazides and triazolophthalazine derivatives (Yoya et al., 2009; De et al., 2011b) and evaluated their anti-TB efficacy **(Scheme 10)**. While 4-alkoxy substitutions were introduced to control the required lipophilicity following Lipinski's rules (Lipinski et al., 1997), their coupling partners were suitably chosen either to mimic biological intermediates or to modify any existing drug. Accordingly, various 4-alkoxycinnamic acids were coupled with *N*-acetylcysteamine (**62**) to afford the corresponding thioesters (**62a,e,f**) thereby mimicking the enoylacyl-ACP intermediate involved in the *M.tb*. fatty acid synthase II (FASII) cycle, an essential step towards mycolic acid (C26-C56 fatty acids) biosynthesis. Mycolic acids are essential components of bacterial cell wall and notably, isoniazid is known to inhibit InhA (enoylacylreductase A; involved in FAS II cycle) thereby inhibiting the mycolic acid biosynthesis. The major advantage of FAS II-cycle as drug target is that it is an exclusive feature of prokaryotes. However, the synthesized thioesters (**62a,e,f**) showed poor anti-TB activities against *M.tb.* H37Rv, possibly due to the weak C-S bond energy which makes these molecules labile under physiological conditions. No amide derivatives (**63a,e, 64a,b,d,f; 65a,e,f**) showed good biological activity except (*E*)-*N*-(2-acetamidoethyl)-3-(4 geranyloxyphenyl)acrylamide (**63f**) (MIC = 0.24 µM, *vs* INH; MIC = 0.6 µM). Unfortunately, they (**63a,e, 64a,b,d,f; 65a,e,f**) have poor cytotoxicity profile. To alleviate the concern for the proteolytic instability, we thus prepared a series of cinnamoylhydrazides (**66a-f**). All six 4 alkoxycinnamoyl isonicotinyl hydrazides (**66a-f**) showed good MIC and their cytotoxicity profile (IC50 ranging between 43-256 μM on THP-1 cell line) were very much encouraging.

cytoplasmic membrane (Roosenberg et al., 2000). The mycobactin so-associated with iron either remains in the cell wall as an iron storage pool or is released into the cell by a mycobactin reductase. Therefore, the sequestration of the available iron into a form, which cannot be processed by *M.tb.* may be an alternative therapeutic way. The success of this approach relies on the understanding of the molecular recognition events involved in mycobacterial iron transport. In that context, the authors synthesized different iron chelators containing α,β-unsaturated hydroxamic acid motifs appended to a citric acid platform such as Nannochelin A (**56b**) and compared their activities with the corresponding *trans*-octenoyl derivative (**56a**). As shown in **Scheme 8**, the synthesis starts from diamines (**52a,b**) with a three-steps sequence to afford, after monoprotection, *N*benzoylation and *N*-acylation with *trans*-cinnamoyl chloride, the dissymetric aminocompounds **53a,b**. **53a,b** were then treated with a 10% NH4OH solution in methanol solution at 0 °C to deprotect the hydroxamic acid and the resulting derivatives were treated with trifluoroacetic acid (TFA) to produce the TFA salts **54a,b**. Finally, the condensation of **54a,b** with the activated Boc-protected citric acid (**55**) in 1,4-dioxane followed by TFA treatment gave the desired chelators **56a,b.** Notably, molecules that provided significantly higher growth index (GI) values than the native chelator **51** were identified as superior growth stimulants and more efficacious iron delivery agents. The systems containing longer tethers gave higher GI values (e.g., GI = 0.76 for **56a** *vs* 1.5 for **56b**). It was envisaged by the authors that the longer tether allows for a more conformationally flexible ligand to properly coordinate to iron thus providing an increase in hydrophobicity. However, the authors identified **56b** as a superior growth stimulant and a more efficacious iron delivery agent. Such ligands, which offer regulation of the initial iron delivery step, provide the opportunity to compare the iron transport mechanisms of both native and genetically modified mycobacteria.

Scheme 8. Synthesis of Nannochelin A

cytoplasmic membrane (Roosenberg et al., 2000). The mycobactin so-associated with iron either remains in the cell wall as an iron storage pool or is released into the cell by a mycobactin reductase. Therefore, the sequestration of the available iron into a form, which cannot be processed by *M.tb.* may be an alternative therapeutic way. The success of this approach relies on the understanding of the molecular recognition events involved in mycobacterial iron transport. In that context, the authors synthesized different iron chelators containing α,β-unsaturated hydroxamic acid motifs appended to a citric acid platform such as Nannochelin A (**56b**) and compared their activities with the corresponding *trans*-octenoyl derivative (**56a**). As shown in **Scheme 8**, the synthesis starts from diamines (**52a,b**) with a three-steps sequence to afford, after monoprotection, *N*benzoylation and *N*-acylation with *trans*-cinnamoyl chloride, the dissymetric aminocompounds **53a,b**. **53a,b** were then treated with a 10% NH4OH solution in methanol solution at 0 °C to deprotect the hydroxamic acid and the resulting derivatives were treated with trifluoroacetic acid (TFA) to produce the TFA salts **54a,b**. Finally, the condensation of **54a,b** with the activated Boc-protected citric acid (**55**) in 1,4-dioxane followed by TFA treatment gave the desired chelators **56a,b.** Notably, molecules that provided significantly higher growth index (GI) values than the native chelator **51** were identified as superior growth stimulants and more efficacious iron delivery agents. The systems containing longer tethers gave higher GI values (e.g., GI = 0.76 for **56a** *vs* 1.5 for **56b**). It was envisaged by the authors that the longer tether allows for a more conformationally flexible ligand to properly coordinate to iron thus providing an increase in hydrophobicity. However, the authors identified **56b** as a superior growth stimulant and a more efficacious iron delivery agent. Such ligands, which offer regulation of the initial iron delivery step, provide the opportunity to compare the iron transport

mechanisms of both native and genetically modified mycobacteria.

Scheme 8. Synthesis of Nannochelin A

#### **2.3.6 Cinnamic acid hydrazide, thioester and other derivatives**

*trans*-Cinnamic acid hydrazide derivatives were presented as potential antituberculosis agents (Carvalho et al. 2008). The authors designed and explored the introduction of the *trans*-cinnamic moiety into isoniazid (**11**) core structure to ameliorate its activity. Isosteric substitution of the pyridine ring of **11** was also investigated by these authors. The synthetic route (**Scheme 9**) used for the preparation of the target compounds is rapid and relies on the formation/use of *p*-nitrophenyl esters (**58a-d**) as activated forms of cinnamic acid derivatives (**Scheme 9**). These stable intermediates (**58a-d**) were prepared by treating the appropriate cinnamic acid (**57a-d**) with thionyl chloride in the presence of 4-nitro-phenol. The target hydrazides (**11a-d**, **59a-d**; **Scheme 9**) were then obtained in good yields by coupling the so-formed activated acids (**58a-d**) with either acylhydrazide **11** or **59**. The anti-TB activities of these compounds were assessed against *M.tb*. Almost all of the isonicotinic derivatives **11a-d** were sensitive in the minimum concentration tested (MIC = 3.12 µg/mL). Nevertheless, all benzoic acid derivatives **59a-d** were much less active, thus reinforcing the pharmacophoric contribution of the isonicotinic moiety. Importantly, the authors identified that the 4-methoxycinnamic derivatives promote the better activity.

Scheme 9. Synthetic route for the preparation of the cinnamoyl hydrazides

In our recent effort, we have synthesized some 4-alkoxycinnamic acid thioesters, amides, hydrazides and triazolophthalazine derivatives (Yoya et al., 2009; De et al., 2011b) and evaluated their anti-TB efficacy **(Scheme 10)**. While 4-alkoxy substitutions were introduced to control the required lipophilicity following Lipinski's rules (Lipinski et al., 1997), their coupling partners were suitably chosen either to mimic biological intermediates or to modify any existing drug. Accordingly, various 4-alkoxycinnamic acids were coupled with *N*-acetylcysteamine (**62**) to afford the corresponding thioesters (**62a,e,f**) thereby mimicking the enoylacyl-ACP intermediate involved in the *M.tb*. fatty acid synthase II (FASII) cycle, an essential step towards mycolic acid (C26-C56 fatty acids) biosynthesis. Mycolic acids are essential components of bacterial cell wall and notably, isoniazid is known to inhibit InhA (enoylacylreductase A; involved in FAS II cycle) thereby inhibiting the mycolic acid biosynthesis. The major advantage of FAS II-cycle as drug target is that it is an exclusive feature of prokaryotes. However, the synthesized thioesters (**62a,e,f**) showed poor anti-TB activities against *M.tb.* H37Rv, possibly due to the weak C-S bond energy which makes these molecules labile under physiological conditions. No amide derivatives (**63a,e, 64a,b,d,f; 65a,e,f**) showed good biological activity except (*E*)-*N*-(2-acetamidoethyl)-3-(4 geranyloxyphenyl)acrylamide (**63f**) (MIC = 0.24 µM, *vs* INH; MIC = 0.6 µM). Unfortunately, they (**63a,e, 64a,b,d,f; 65a,e,f**) have poor cytotoxicity profile. To alleviate the concern for the proteolytic instability, we thus prepared a series of cinnamoylhydrazides (**66a-f**). All six 4 alkoxycinnamoyl isonicotinyl hydrazides (**66a-f**) showed good MIC and their cytotoxicity profile (IC50 ranging between 43-256 μM on THP-1 cell line) were very much encouraging.

Cinnamic Derivatives in Tuberculosis 349

also evaluated. Significantly, the MICs of the compounds (**69a-f**) were found to be poorer compared to **68a-f**. In regard to the difference in activities between the enoyl and cyclopropyl series, a plausible explanation could be the respective Michael acceptor ability. From a chemical point of view, 4-OCF3 derivatives are expected to show better inhibitory activities compared to their 4-OCH3 analogues. However, this is not the case as **66a** has a 4 fold better activity (MIC = 0.3 µM) compared to **66b** (MIC = 1.1 µM) and similarly **68a** (MIC = 53 µM) exhibits approximately 15-fold activity better than **68b** (702 µM). In view of these results, it was suggested that the Michael addition may not be the mode of action of these compounds. This view was also supported by the fact that mycobacterial lip B prefers to form thioester intermediate with deca-2-enoic acid during mycolic acid biosynthesis unlike

Cinnamaldehyde (**70**), also biosynthesized starting from phenylalanine (**9**) in the process of lignin biosynthesis, occurs naturally in the bark of cinnamon trees and other species of the genus *Cinnamomum*. Owing to its typical odor and low toxicity to human exposure, cinnamaldehyde is used as food flavoring agent. It is also used as a fungicide, insecticide for mosquito larvae (Cheng et al*.,* 2004) and has shown inhibitory activities towards proliferation, invasion and tumor growth in a murin A375 model of human melanoma (Cabello et al*.,* 2009). Importantly, **70** and its derivatives have shown enormous potential as antimicrobial agents. For example, cinnamaldehyde is known to inhibit *E. coli* and *Salmonella typhimurium* growth (Helander et al*.,* 1998). Its carbonyl group has affinity for proteins, preventing the action of decarboxylase amino acids on *E. aerogenes* (Wendakoon & Sakaguchi, 1995). From a chemical standpoint, worth precising is that **70** so as its 3 phenylacrylaldehydic congeners offer three main reactive sites: substitution on the phenyl ring, addition on the α,β-unsaturation and reactions of the aldehyde functionality. The α,βunsaturated carbonyl moiety can be considered as a Michael acceptor (Chew et al.*,* 2010),

The growth of *M. avium* subsp. paratuberculosis is inhibited by cinnamaldehyde (**70**) with a MIC of 25.9 μg/mL (Wong et al*.,* 2008). Importantly, the authors suggest that the mechanism of antimicrobial activity of naturally occurring compounds such as cinnamaldehyde is specific rather than nonspecific since it is concentration dependent (Friedman et al*.,* 2002). Possible modes of action include disruption of cell membranes, inhibition of essential enzymes, chelation of essential trace elements (such as iron), and targeting of cell membranes. Cinnamaldehyde (**70**) is also known to inhibit the bacterial cell division protein FtsZ (Domadia et al*.,* 2007). FtsZ, a prokaryotic homolog of tubulin, regulates cell by assembling into the macromolecular structure called Z-ring at the site of cell division (Romberg & Levin, 2003). While cinnamaldehyde (**70**) proves to decrease the *in vitro* assembly reaction and bundling of FtsZ, **70** was also found to perturb the Z-ring morphology *in vivo* and to reduce the frequency of the Z ring per unit cell length of *Escherichia coli*. In addition, GTP-dependent FtsZ polymerization is inhibited by **70**, cinnamaldehyde (**70**) inhibiting the rate of GTP hydrolysis and binding FtsZ with an affinity constant of 1.0 ± 0.2 μM−1. Isothermal titration calorimetry revealed that the binding of **70** to

*E.coli* lipB which forms a thioether *via* Michael addition (Ma et al.; 2006).

**3. Cinnamaldehyde derivatives as anti-TB agents** 

which is often employed in the design of drugs (Ahn & Sok, 1996).

**3.1 Cinnamaldehyde** 

Further, the radio-thin-layer chromatography analysis of **52e**, when introduced to the broth culture of *M.tb.,* revealed that this class of molecules inhibit the mycolic acid biosynthesis. Two representative INH-derivatives **66a** (same as **11b** in **Scheme 9**) and **66e** were tested on MYC5165, a *M.tb.* strain mutated in InhA and 1400 a *M.tb.* strain mutated in *katG*. The inhibitory activities of **66a** (MIC = 16 µM: MYC5165; 320 µM: 1400) and **66e** (MIC = 27 µM: MYC5165; 68 µM: 1400) were found to follow similar trends as that of INH (MIC = 18 µM: MYC5165; 729 µM: 1400) itself**,** thus not allowing at the moment to propose these compounds as isoniazid prodrugs or not. In order to explore the influence of other hydrazides, 1-hydrazinophthalazine hydrochloride **67**, an antihypertensive drug (Schroeder, 1952; Silas et al., 1982) of moderate potency, was coupled with acids **60a-f** in the presence of EDC.HCl, HOBt and triethylamine to afford phthalazinohydrazides (**67a-f**). For the family of 1-phthalazinohydrazides (**67a-f**), MIC results were moderate but the trend of cytotoxic behaviour was not acceptable. Under different experimental conditions, coupling of acids (**60a-f**) with **67** in acetonitrile under reflux furnished the corresponding 3-(4-alkoxystyryl)- [1,2,4]triazolo[3,4-]phthalazines (**68a-f**). Interestingly, the combination of isopentenyl-side chain as 4-alkoxy substituent with triazolophthalazine (**68e**), showed excellent antitubercular potency (MIC = 1.4 µM), in comparison with other derivatives in the series (**68a-f**), and more importantly, with good cytotoxicity (IC50 = 449 µM on THP-1 cell line) and selectivity index (SI = 320). Finally, to our great delight, compound **68e** showed 100-fold better *in vitro* activity against MYC5165 strain (**68e**; MIC = 0.2 M) and 1800-fold better activity against 1400 strain (**68e**; MIC= 0.4 M) compared to INH.

Scheme 10. Synthetic route for the preparation of different cinnamoyl derivatives

Further, the radio-thin-layer chromatography analysis revealed that compound **68e** does not inhibit mycolic acid biosynthesis signifying a different mode of action than INH. In order to explore the importance of the enoyl-acyl backbone, the double bond was replaced by bioisosteric cyclopropyl moiety. Thus, 3-[2-(4-alkoxyphenyl)cyclopropyl]-[1,2,4]triazolo[3,4 α]phthalazine (**69a-f**; racemates) were synthesized and their *in-vitro* anti-TB activities were

Further, the radio-thin-layer chromatography analysis of **52e**, when introduced to the broth culture of *M.tb.,* revealed that this class of molecules inhibit the mycolic acid biosynthesis. Two representative INH-derivatives **66a** (same as **11b** in **Scheme 9**) and **66e** were tested on MYC5165, a *M.tb.* strain mutated in InhA and 1400 a *M.tb.* strain mutated in *katG*. The inhibitory activities of **66a** (MIC = 16 µM: MYC5165; 320 µM: 1400) and **66e** (MIC = 27 µM: MYC5165; 68 µM: 1400) were found to follow similar trends as that of INH (MIC = 18 µM: MYC5165; 729 µM: 1400) itself**,** thus not allowing at the moment to propose these compounds as isoniazid prodrugs or not. In order to explore the influence of other hydrazides, 1-hydrazinophthalazine hydrochloride **67**, an antihypertensive drug (Schroeder, 1952; Silas et al., 1982) of moderate potency, was coupled with acids **60a-f** in the presence of EDC.HCl, HOBt and triethylamine to afford phthalazinohydrazides (**67a-f**). For the family of 1-phthalazinohydrazides (**67a-f**), MIC results were moderate but the trend of cytotoxic behaviour was not acceptable. Under different experimental conditions, coupling of acids (**60a-f**) with **67** in acetonitrile under reflux furnished the corresponding 3-(4-alkoxystyryl)- [1,2,4]triazolo[3,4-]phthalazines (**68a-f**). Interestingly, the combination of isopentenyl-side chain as 4-alkoxy substituent with triazolophthalazine (**68e**), showed excellent antitubercular potency (MIC = 1.4 µM), in comparison with other derivatives in the series (**68a-f**), and more importantly, with good cytotoxicity (IC50 = 449 µM on THP-1 cell line) and selectivity index (SI = 320). Finally, to our great delight, compound **68e** showed 100-fold better *in vitro* activity against MYC5165 strain (**68e**; MIC = 0.2 M) and 1800-fold better

activity against 1400 strain (**68e**; MIC= 0.4 M) compared to INH.

Scheme 10. Synthetic route for the preparation of different cinnamoyl derivatives

Further, the radio-thin-layer chromatography analysis revealed that compound **68e** does not inhibit mycolic acid biosynthesis signifying a different mode of action than INH. In order to explore the importance of the enoyl-acyl backbone, the double bond was replaced by bioisosteric cyclopropyl moiety. Thus, 3-[2-(4-alkoxyphenyl)cyclopropyl]-[1,2,4]triazolo[3,4 α]phthalazine (**69a-f**; racemates) were synthesized and their *in-vitro* anti-TB activities were also evaluated. Significantly, the MICs of the compounds (**69a-f**) were found to be poorer compared to **68a-f**. In regard to the difference in activities between the enoyl and cyclopropyl series, a plausible explanation could be the respective Michael acceptor ability. From a chemical point of view, 4-OCF3 derivatives are expected to show better inhibitory activities compared to their 4-OCH3 analogues. However, this is not the case as **66a** has a 4 fold better activity (MIC = 0.3 µM) compared to **66b** (MIC = 1.1 µM) and similarly **68a** (MIC = 53 µM) exhibits approximately 15-fold activity better than **68b** (702 µM). In view of these results, it was suggested that the Michael addition may not be the mode of action of these compounds. This view was also supported by the fact that mycobacterial lip B prefers to form thioester intermediate with deca-2-enoic acid during mycolic acid biosynthesis unlike *E.coli* lipB which forms a thioether *via* Michael addition (Ma et al.; 2006).

## **3. Cinnamaldehyde derivatives as anti-TB agents**

Cinnamaldehyde (**70**), also biosynthesized starting from phenylalanine (**9**) in the process of lignin biosynthesis, occurs naturally in the bark of cinnamon trees and other species of the genus *Cinnamomum*. Owing to its typical odor and low toxicity to human exposure, cinnamaldehyde is used as food flavoring agent. It is also used as a fungicide, insecticide for mosquito larvae (Cheng et al*.,* 2004) and has shown inhibitory activities towards proliferation, invasion and tumor growth in a murin A375 model of human melanoma (Cabello et al*.,* 2009). Importantly, **70** and its derivatives have shown enormous potential as antimicrobial agents. For example, cinnamaldehyde is known to inhibit *E. coli* and *Salmonella typhimurium* growth (Helander et al*.,* 1998). Its carbonyl group has affinity for proteins, preventing the action of decarboxylase amino acids on *E. aerogenes* (Wendakoon & Sakaguchi, 1995). From a chemical standpoint, worth precising is that **70** so as its 3 phenylacrylaldehydic congeners offer three main reactive sites: substitution on the phenyl ring, addition on the α,β-unsaturation and reactions of the aldehyde functionality. The α,βunsaturated carbonyl moiety can be considered as a Michael acceptor (Chew et al.*,* 2010), which is often employed in the design of drugs (Ahn & Sok, 1996).

#### **3.1 Cinnamaldehyde**

The growth of *M. avium* subsp. paratuberculosis is inhibited by cinnamaldehyde (**70**) with a MIC of 25.9 μg/mL (Wong et al*.,* 2008). Importantly, the authors suggest that the mechanism of antimicrobial activity of naturally occurring compounds such as cinnamaldehyde is specific rather than nonspecific since it is concentration dependent (Friedman et al*.,* 2002). Possible modes of action include disruption of cell membranes, inhibition of essential enzymes, chelation of essential trace elements (such as iron), and targeting of cell membranes. Cinnamaldehyde (**70**) is also known to inhibit the bacterial cell division protein FtsZ (Domadia et al*.,* 2007). FtsZ, a prokaryotic homolog of tubulin, regulates cell by assembling into the macromolecular structure called Z-ring at the site of cell division (Romberg & Levin, 2003). While cinnamaldehyde (**70**) proves to decrease the *in vitro* assembly reaction and bundling of FtsZ, **70** was also found to perturb the Z-ring morphology *in vivo* and to reduce the frequency of the Z ring per unit cell length of *Escherichia coli*. In addition, GTP-dependent FtsZ polymerization is inhibited by **70**, cinnamaldehyde (**70**) inhibiting the rate of GTP hydrolysis and binding FtsZ with an affinity constant of 1.0 ± 0.2 μM−1. Isothermal titration calorimetry revealed that the binding of **70** to

Cinnamic Derivatives in Tuberculosis 351

Scheme 13. Different hydrazone derivatives synthesized from cinnamaldehyde

(**86**) which showed anti-TB activity (MIC = 25 μg/mL).

**3.3 Cinnamaldehyde-derived metal complexes** 

Scheme 14. Synthesis of a quinaxoline-derived cinnamaldehyde hydrazone

A new series of copper(II) and zinc(II) complexes has been designed and synthesized using a new type of Schiff base (**89a-h**) derived from the reaction of 3-(3-phenyl-allylidene) pentane-2,4-dione (**88**) with *para*-substituted aniline and benzene-1,2-dithiol (**90**) (**Scheme 15**) (Raman et al., 2010). The intermediate **88** was first obtained by aldol condensation between **70** and acetylacetone (**87**) using piperidine as base. The minimum inhibitory concentrations of the complexes have also been investigated against *M.tb.* strain H37Rv. The lowest MIC values were obtained for -NO2 group containing complexes (**91g**; MIC = 2.9

Several other hydrazone derivatives (**79a-e**) of cinnamaldehyde were made and their antiTB efficacy was also tested(**Scheme 13**) (Abdel-Aal et al. 2009). Among them, compound **79d**, arising from cinnamaldehyde (**70**) and isoniazid (**11**), showed maximum anti-TB activity (at 50 µg/mL) with the same MIC of the reference drug isoniazid (INH, 12.5 µg/mL).The synthetic hydrazone (**86**), exhibiting anti-TB activity (MIC = 25 μg/mL), was recently reported by Rao *et al.* (**Scheme 14**) (Rao et al*.,* 2010). The authors synthesized **86** through 3-methyl-2-oxoquinoxalin (**82**) that is easily obtainable from *ortho*diaminobenzene (**80**) by refluxing in ethanol along with acetylacetic acid (**81**). Treatment of **82** with 2-chloro ethylacetate (**83**) in the presence of K2CO3 in DMF gave the corresponding ethyl 2-(3-methyl-2-oxoquinoxalin-1(2*H*)-yl)acetate (**84**) which was converted to 2-(3-methyl-2-oxoquinoxalin-1(2*H*)-yl)acetohydrazide (**85**) by refluxing in ethanol in the presence of hydrazine hydrate (**Scheme 14**). Compound **85** was then coupled with **70** in DMF in the presence of acetic acid to furnish the desired hydrazone

FtsZ is driven by favorable enthalpic interactions. This signifies that **70** binds FtsZ, perturbs the cytokinetic Z-ring formation and inhibits its assembly dynamics. The authors suggested that **70**, a small molecule of plant origin, is a potential lead compound that can be developed as an anti-FtsZ agent towards drug design.

#### **3.2 Cinnamaldehyde-derived hydrazones**

The synthesis and antimycobacterial efficacy of a new class of styryl derivatives (**74a-c**) were reported (**Scheme 11**) (Biava et al*.,* 1997). The desired styryl derivatives (**74a-c**) were prepared in a three-steps sequence that begins by the reduction of the starting nitro compounds (**71a-c**) furnishing *ortho-*, *meta-* or *para*-aminotoluidines possessing either an imidazole, pyrazine or morpholine frame (**72a-c**). The so-obtained compounds (**72a-c**) were then coupled under reductive amination conditions (**Scheme 11**) with cinnamaldehyde (**70**) to afford the toluidine-styryl derivatives (**74a-c**). Among all synthetized compounds, derivatives **73c** (R=Imidazole) and **74a-c** (R=Imidazole) were the most active against five different *M.tb.* strains with MIC values ranging between 1 to 64 µg/mL.

Scheme 11. Synthesis of toluidine derivatives

In 2010, a series of 5-(4-isopropylthiazol-2-yl)-4-((*E*)-((*E*)-3-phenylallylidene)amino)-4*H*-1,2,4-triazole-3-thiol (**78**; **Scheme 12**) was synthesized (Kumar *et al.* 2010). 4- Isopropylthiazol-2-carbahydrazide **76** was converted into the corresponding dithiocarbazinate, which upon cyclization with hydrazine hydrate yields 4-amino-5-(4 isopropyl-1,3-thiazol-2-yl)-4*H*-1,2,4-triazole-3-thiol (**77**). The triazole (**77**) was condensed with **70** in the presence of catalytic amount of H2SO4 in refluxing ethanol to afford **78**. Synthesized compound **78** was evaluated (Shiradkar et al. 2007; Joshi et al. 2008) for its preliminary cytotoxicity and antitubercular activity against *M.tb*. H37Rv strain by broth dilution assay method and showed a promising activity (MIC = 4 µg/mL).

Scheme 12. Synthesis of 2-substituted -5-[isopropylthiazole] clubbed 1,2,4-triazole

FtsZ is driven by favorable enthalpic interactions. This signifies that **70** binds FtsZ, perturbs the cytokinetic Z-ring formation and inhibits its assembly dynamics. The authors suggested that **70**, a small molecule of plant origin, is a potential lead compound that can be developed

The synthesis and antimycobacterial efficacy of a new class of styryl derivatives (**74a-c**) were reported (**Scheme 11**) (Biava et al*.,* 1997). The desired styryl derivatives (**74a-c**) were prepared in a three-steps sequence that begins by the reduction of the starting nitro compounds (**71a-c**) furnishing *ortho-*, *meta-* or *para*-aminotoluidines possessing either an imidazole, pyrazine or morpholine frame (**72a-c**). The so-obtained compounds (**72a-c**) were then coupled under reductive amination conditions (**Scheme 11**) with cinnamaldehyde (**70**) to afford the toluidine-styryl derivatives (**74a-c**). Among all synthetized compounds, derivatives **73c** (R=Imidazole) and **74a-c** (R=Imidazole) were the most active against five

In 2010, a series of 5-(4-isopropylthiazol-2-yl)-4-((*E*)-((*E*)-3-phenylallylidene)amino)-4*H*-1,2,4-triazole-3-thiol (**78**; **Scheme 12**) was synthesized (Kumar *et al.* 2010). 4- Isopropylthiazol-2-carbahydrazide **76** was converted into the corresponding dithiocarbazinate, which upon cyclization with hydrazine hydrate yields 4-amino-5-(4 isopropyl-1,3-thiazol-2-yl)-4*H*-1,2,4-triazole-3-thiol (**77**). The triazole (**77**) was condensed with **70** in the presence of catalytic amount of H2SO4 in refluxing ethanol to afford **78**. Synthesized compound **78** was evaluated (Shiradkar et al. 2007; Joshi et al. 2008) for its preliminary cytotoxicity and antitubercular activity against *M.tb*. H37Rv strain by broth

different *M.tb.* strains with MIC values ranging between 1 to 64 µg/mL.

dilution assay method and showed a promising activity (MIC = 4 µg/mL).

Scheme 12. Synthesis of 2-substituted -5-[isopropylthiazole] clubbed 1,2,4-triazole

as an anti-FtsZ agent towards drug design.

**3.2 Cinnamaldehyde-derived hydrazones** 

Scheme 11. Synthesis of toluidine derivatives

Scheme 13. Different hydrazone derivatives synthesized from cinnamaldehyde

Several other hydrazone derivatives (**79a-e**) of cinnamaldehyde were made and their antiTB efficacy was also tested(**Scheme 13**) (Abdel-Aal et al. 2009). Among them, compound **79d**, arising from cinnamaldehyde (**70**) and isoniazid (**11**), showed maximum anti-TB activity (at 50 µg/mL) with the same MIC of the reference drug isoniazid (INH, 12.5 µg/mL).The synthetic hydrazone (**86**), exhibiting anti-TB activity (MIC = 25 μg/mL), was recently reported by Rao *et al.* (**Scheme 14**) (Rao et al*.,* 2010). The authors synthesized **86** through 3-methyl-2-oxoquinoxalin (**82**) that is easily obtainable from *ortho*diaminobenzene (**80**) by refluxing in ethanol along with acetylacetic acid (**81**). Treatment of **82** with 2-chloro ethylacetate (**83**) in the presence of K2CO3 in DMF gave the corresponding ethyl 2-(3-methyl-2-oxoquinoxalin-1(2*H*)-yl)acetate (**84**) which was converted to 2-(3-methyl-2-oxoquinoxalin-1(2*H*)-yl)acetohydrazide (**85**) by refluxing in ethanol in the presence of hydrazine hydrate (**Scheme 14**). Compound **85** was then coupled with **70** in DMF in the presence of acetic acid to furnish the desired hydrazone (**86**) which showed anti-TB activity (MIC = 25 μg/mL).

Scheme 14. Synthesis of a quinaxoline-derived cinnamaldehyde hydrazone

#### **3.3 Cinnamaldehyde-derived metal complexes**

A new series of copper(II) and zinc(II) complexes has been designed and synthesized using a new type of Schiff base (**89a-h**) derived from the reaction of 3-(3-phenyl-allylidene) pentane-2,4-dione (**88**) with *para*-substituted aniline and benzene-1,2-dithiol (**90**) (**Scheme 15**) (Raman et al., 2010). The intermediate **88** was first obtained by aldol condensation between **70** and acetylacetone (**87**) using piperidine as base. The minimum inhibitory concentrations of the complexes have also been investigated against *M.tb.* strain H37Rv. The lowest MIC values were obtained for -NO2 group containing complexes (**91g**; MIC = 2.9

Cinnamic Derivatives in Tuberculosis 353

for growth of *M.tb.* in human macrophages (Bach et al*.,* 2008). In the search for lead compounds, a series of 38 chalcones were prepared by aldol condensation between aldehydes and acetophenones and five of the so-prepared compounds (**95-99**) presented moderate to good activities (**Scheme 16**). The structure–activity analysis revealed that the predominant factor for the activity is the molecular planarity and/or hydrophobicity and the nature of the substituents. Later on, the molecular recognition of these inhibitors on PtpA was investigated through molecular modeling, these investigations revealing that the binding and the inhibitory activity of the chalcones are predominantly governed by the positions of the two methoxyl groups at the B-ring (Mascarello et al*.,* 2010). Besides, the – OMe groups proved to establish key hydrogen bonds with the amino acid residues Arg17, His49 and Thr12 in the active site of PtpA while the 2-naphthyl A-ring undergoes л-stacking interaction with the Trp48 residue. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel

A series of *N*΄-nicotinoyl-3-(4΄-hydroxy-3΄-methylphenyl)-5-(substituted phenyl)-2 pyrazolines (**102a-d**) were synthesized by the reaction between isoniazid (INH; **11**) and chalcones (**101a-d**) and were tested for their antimycobacterial activity *in vitro* against *M.tb*. H37Rv and INH-resistant *M.tb.* (INHR- *M.tb*.) strains using the agar dilution method(**Scheme 17**) (Shaharyar et al. 2006). Among the synthesized compounds, *N*΄ nicotinyl-3-(4΄-hydroxy-3΄-methyl phenyl)-5-(1΄΄-chlorophenyl)-2-pyrazoline (**102d**) was found to be the most active agent against *M.tb*. and INHR- *M.tb*., with minimum inhibitory concentration of 0.26 µM. When compared to INH-compound **102d** was found to be 2.8- and

43.7-fold more active against *M.tb*. H37Rv and INHR-*M.tb*., respectively.

therapeutics.

Fig. 8. Chalcones with antiTB activities

Scheme 16. General strategy for chalcone synthesis

**4.2 Chalcone hydrazone derivatives** 

μg/mL, **91h**; MIC = 3.8 μg/mL) which are more active against H37Rv strain than the other complexes.

Scheme 15. Preparation of Cu(II) and Zn(II) complexes starting from cinnamaldehyde

#### **4. Chalcone derivatives as anti-TB agents**

Chalcones, one of the major classes of natural products with widespread distribution in fruits, vegetables, spices, tea and soya based foodstuffs, are of great interest for their interesting pharmacological activities (Di Carlo et al., 1999). Chalcones, or 1,3-diaryl-2 propen-1-ones, belong to the flavonoid family (Dimmock et al., 1999; Ni et al., 2004). From a structural standpoint, they consist of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl unit (**Fig. 8**). Interestingly, a vast number of naturally occurring chalcones are polyhydroxylated onto both aryl rings conferring them significant radical-quenching properties which have raised interest in using chalcones or chalcone-rich plant extracts as drugs or food preservatives (Nowakowska, 2007). Besides, Chalcones have been reported to possess many useful properties, including anti-inflammatory, antimicrobial, antifungal, antioxidant, cytotoxic, antitumor and anticancer activities (Dimmock et al., 1999; Go, Wu & Liu, 2005).

#### **4.1 Natural and synthesized chalcones**

*M.tb.* , *M. bovis*, *M. kansasii*, *M. xenophii* and *M. marinum* were inhibited by licochalcone A (**92**; MIC = 20 mg /mL), extracted and purified from Chinese licorice roots (**Fig. 8**) (Friis-Møller et al. 2002). Besides, the presence of a halogen substituent on A-ring of 2' hydroxychalcones proved to play a crucial role on anti-TB activity. It has indeed been found that chalcones substituted by a halogen atom at the 3-position demonstrate stronger anti-TB activity than those substituted by a halogen atom at the 2- or 4-position (Lin et al*.,* 2002). In that manner, chalcones **93** and **94** with a 2'-hydroxyl group on B-ring and a 3-chloro- or 3 iodo-group on A-ring showed the strongest activity, with 90-92% inhibition against *M.tb.* H37Rv at a drug concentration of 12.5 mg/mL. The activity of 2΄-hydroxychalcone (61% inhibition) was further enhanced by introducing a chloro (89%) or a methoxy group (78%) at the 4΄-position of B-ring. Nevertheless, introduction of an additional substituent, such as a methoxy, amino, bromo or carboxyl group on B-ring led to a dramatic decrease or a complete loss of activity (Lin et al*.,* 2002). Recently, the activities of some synthetic chalcones were also assayed against *M.tb*. protein tyrosine phosphatase A (PtpA) which is an enzyme associated with *M.tb.* infectivity (Chiaradia et al*.,* 2008). Note that tyrosine phosphatases are secreted by pathogenic bacteria, and MPtpA is an example that was shown to be required

μg/mL, **91h**; MIC = 3.8 μg/mL) which are more active against H37Rv strain than the other

N N

Scheme 15. Preparation of Cu(II) and Zn(II) complexes starting from cinnamaldehyde

Chalcones, one of the major classes of natural products with widespread distribution in fruits, vegetables, spices, tea and soya based foodstuffs, are of great interest for their interesting pharmacological activities (Di Carlo et al., 1999). Chalcones, or 1,3-diaryl-2 propen-1-ones, belong to the flavonoid family (Dimmock et al., 1999; Ni et al., 2004). From a structural standpoint, they consist of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl unit (**Fig. 8**). Interestingly, a vast number of naturally occurring chalcones are polyhydroxylated onto both aryl rings conferring them significant radical-quenching properties which have raised interest in using chalcones or chalcone-rich plant extracts as drugs or food preservatives (Nowakowska, 2007). Besides, Chalcones have been reported to possess many useful properties, including anti-inflammatory, antimicrobial, antifungal, antioxidant, cytotoxic, antitumor and

*M.tb.* , *M. bovis*, *M. kansasii*, *M. xenophii* and *M. marinum* were inhibited by licochalcone A (**92**; MIC = 20 mg /mL), extracted and purified from Chinese licorice roots (**Fig. 8**) (Friis-Møller et al. 2002). Besides, the presence of a halogen substituent on A-ring of 2' hydroxychalcones proved to play a crucial role on anti-TB activity. It has indeed been found that chalcones substituted by a halogen atom at the 3-position demonstrate stronger anti-TB activity than those substituted by a halogen atom at the 2- or 4-position (Lin et al*.,* 2002). In that manner, chalcones **93** and **94** with a 2'-hydroxyl group on B-ring and a 3-chloro- or 3 iodo-group on A-ring showed the strongest activity, with 90-92% inhibition against *M.tb.* H37Rv at a drug concentration of 12.5 mg/mL. The activity of 2΄-hydroxychalcone (61% inhibition) was further enhanced by introducing a chloro (89%) or a methoxy group (78%) at the 4΄-position of B-ring. Nevertheless, introduction of an additional substituent, such as a methoxy, amino, bromo or carboxyl group on B-ring led to a dramatic decrease or a complete loss of activity (Lin et al*.,* 2002). Recently, the activities of some synthetic chalcones were also assayed against *M.tb*. protein tyrosine phosphatase A (PtpA) which is an enzyme associated with *M.tb.* infectivity (Chiaradia et al*.,* 2008). Note that tyrosine phosphatases are secreted by pathogenic bacteria, and MPtpA is an example that was shown to be required

R

R

HS

SH

**90**

MCl2, NaOEt M = Cu, Zn N N

M S S

**91a-h**

R

R M H Cu **91a** H Zn **91b** OH Cu **91c** OH Zn **91d** OMe Cu **91e** OMe Zn **91f** NO2 Cu **91g** NO2 Zn **91h**

R

complexes.

O O

**87**

Piperidine

**70**

O O H2N

**4. Chalcone derivatives as anti-TB agents** 

R

**88 89a-h**

anticancer activities (Dimmock et al., 1999; Go, Wu & Liu, 2005).

**4.1 Natural and synthesized chalcones** 

for growth of *M.tb.* in human macrophages (Bach et al*.,* 2008). In the search for lead compounds, a series of 38 chalcones were prepared by aldol condensation between aldehydes and acetophenones and five of the so-prepared compounds (**95-99**) presented moderate to good activities (**Scheme 16**). The structure–activity analysis revealed that the predominant factor for the activity is the molecular planarity and/or hydrophobicity and the nature of the substituents. Later on, the molecular recognition of these inhibitors on PtpA was investigated through molecular modeling, these investigations revealing that the binding and the inhibitory activity of the chalcones are predominantly governed by the positions of the two methoxyl groups at the B-ring (Mascarello et al*.,* 2010). Besides, the – OMe groups proved to establish key hydrogen bonds with the amino acid residues Arg17, His49 and Thr12 in the active site of PtpA while the 2-naphthyl A-ring undergoes л-stacking interaction with the Trp48 residue. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics.

Fig. 8. Chalcones with antiTB activities

Scheme 16. General strategy for chalcone synthesis

#### **4.2 Chalcone hydrazone derivatives**

A series of *N*΄-nicotinoyl-3-(4΄-hydroxy-3΄-methylphenyl)-5-(substituted phenyl)-2 pyrazolines (**102a-d**) were synthesized by the reaction between isoniazid (INH; **11**) and chalcones (**101a-d**) and were tested for their antimycobacterial activity *in vitro* against *M.tb*. H37Rv and INH-resistant *M.tb.* (INHR- *M.tb*.) strains using the agar dilution method(**Scheme 17**) (Shaharyar et al. 2006). Among the synthesized compounds, *N*΄ nicotinyl-3-(4΄-hydroxy-3΄-methyl phenyl)-5-(1΄΄-chlorophenyl)-2-pyrazoline (**102d**) was found to be the most active agent against *M.tb*. and INHR- *M.tb*., with minimum inhibitory concentration of 0.26 µM. When compared to INH-compound **102d** was found to be 2.8- and 43.7-fold more active against *M.tb*. H37Rv and INHR-*M.tb*., respectively.

Cinnamic Derivatives in Tuberculosis 355

known antituberculosis drug, showed a MIC value of 50 µg/mL under similar assay.

Importantly, a quantitative structure activity relationship (QSARs) methodology has been developed (Sivakumar et al. 2007) for the reported anti-TB activity of chalcones, chalconelike compounds, flavones and flavanones using a statistical technique called genetic function approximation (GFA). The generated equations in each model were analyzed, for both the goodness of fit and predictive capability. The analysis also points out to the importance of the hydrogen, PMI-mag and HOMO bond donors. The study indicated that the reported compounds are more lipophilic in nature and hence, as expected, exhibit good activity since *M.tb.* has a high concentration of lipid layer. These theoretical models deserve to be explored further to design potent, newer compounds having better anti-TB activity.

Cinnamic acids, cinnamaldehydes and chalcones are unique as drug candidates in tuberculosis. Natural cinnamic-based substances such as ethyl- (**2**) and benzyl- (**4**) cinnamates not only have anti-TB activities, they are traditional medicines for hypertension as well. Cinnamaldehyde and Licochalcone A also have good potentials as anti-TB agents. Sharing a common α,β-unsaturated carbonyl functionality, these molecules offer Michaelacceptor properties, particularly to the glutathione (GSH) and cystine residues. Although, mycobacteria, unlike *E.coli*, do not prefer the formation of Michael-adduct, the presence of the cinnamic moiety certainly increased the anti-TB efficacy on several occasions. Importantly, the replacement of the double bond with an isosteric cyclopropyl ring decreased the anti-TB efficiency of the triazolophthalazines. On the other hand, introduction of cinnamoyl moiety to isoniazid did not significantly alter the trend of biological activity or the mode of action. These observations indicate that the anti-TB activity depends not only on the α,β-unsaturation but also on the functionalization of the carbonyl part of the cinnamoyl derivatives. Several hydrazone derivatives of cinnamaldehyde and chalcones have notable anti-TB activities. Importantly, substituents at the benzene ring of the cinnamic acids also play a crucial role in the biological activities. Notably, Isoprenyloxy cinnamoyltriazolophthalazine derivative (**68e**) and chalcone derived pyrazoline derivative (**102d**)

Moreover, these molecules were nontoxic against VERO and MBMDM cell lines.

Scheme 19. Synthesis of substituted quinolinyl chalcones

**4.4 Physico-chemical study** 

**5. Conclusion** 

Scheme 17. Synthesis and anti-TB activities of chalcone-derived pyrazoline compounds

#### **4.3 Chalcones with substitutions at the aromatic ring**

A series of acetylenic chalcones were evaluated for antituberculosis activity (**Scheme 18**) (Hans et al., 2010). The acetylenic functionality not only serves as a site for further chemical diversification but is also of great interest in medicinal chemistry and the pharmaceutical industry. Moreover, it functions as a key pharmacophoric unit in acetylenic antibiotics (Maretina & Trofimov, 2006) and its presence in anticancer (Siddiq & Dembitsky, 2008) and antitubercular (Deng et al. 2008) agents is noteworthy. From a synthetic standpoint, hydroxyacetophenones (**103a,b**) were treated with propargyl bromide (**104**) in the presence of K2CO3 in DMF to afford the respective propargyloxyacetophenones (**105a,b**) that were then treated with methoxybenzaldehydes (**106,107**) under basic conditions to provide the chalcones (**108a,b**) featuring the desired propargyloxy moiety. Most compounds were more active against non-replicating (MABA) than replicating (LORA) cultures of *M.tb.* H37Rv, an unusual pattern with respect to existing anti-TB agents.

Scheme 18. Synthesis and anti-TB activities of acetylenic chalcones

The introduction of a quinoline moiety to chalcones as aromatic substituent was envisaged as an interesting way of designing new anti-TB agents. In that manner, a series of substituted quinolinyl chalcones (**113**, **114**) was synthesized under basic conditions and evaluated for their *in vitro* anti-TB activity against *M.tb.* H37Rv (**Scheme 19**) (Sharma et al*.,* 2009). The structure–activity relationship analysis revealed that different physicochemical and structural requirements are needed for anti-TB activity. Two compounds **113** and **114** have shown anti-TB activity at MIC 3.12 µg/mL. By comparison, pyrazinamide (**115**), a

Scheme 17. Synthesis and anti-TB activities of chalcone-derived pyrazoline compounds

A series of acetylenic chalcones were evaluated for antituberculosis activity (**Scheme 18**) (Hans et al., 2010). The acetylenic functionality not only serves as a site for further chemical diversification but is also of great interest in medicinal chemistry and the pharmaceutical industry. Moreover, it functions as a key pharmacophoric unit in acetylenic antibiotics (Maretina & Trofimov, 2006) and its presence in anticancer (Siddiq & Dembitsky, 2008) and antitubercular (Deng et al. 2008) agents is noteworthy. From a synthetic standpoint, hydroxyacetophenones (**103a,b**) were treated with propargyl bromide (**104**) in the presence of K2CO3 in DMF to afford the respective propargyloxyacetophenones (**105a,b**) that were then treated with methoxybenzaldehydes (**106,107**) under basic conditions to provide the chalcones (**108a,b**) featuring the desired propargyloxy moiety. Most compounds were more active against non-replicating (MABA) than replicating (LORA) cultures of *M.tb.* H37Rv, an

**4.3 Chalcones with substitutions at the aromatic ring** 

unusual pattern with respect to existing anti-TB agents.

Scheme 18. Synthesis and anti-TB activities of acetylenic chalcones

The introduction of a quinoline moiety to chalcones as aromatic substituent was envisaged as an interesting way of designing new anti-TB agents. In that manner, a series of substituted quinolinyl chalcones (**113**, **114**) was synthesized under basic conditions and evaluated for their *in vitro* anti-TB activity against *M.tb.* H37Rv (**Scheme 19**) (Sharma et al*.,* 2009). The structure–activity relationship analysis revealed that different physicochemical and structural requirements are needed for anti-TB activity. Two compounds **113** and **114** have shown anti-TB activity at MIC 3.12 µg/mL. By comparison, pyrazinamide (**115**), a known antituberculosis drug, showed a MIC value of 50 µg/mL under similar assay. Moreover, these molecules were nontoxic against VERO and MBMDM cell lines.

Scheme 19. Synthesis of substituted quinolinyl chalcones

#### **4.4 Physico-chemical study**

Importantly, a quantitative structure activity relationship (QSARs) methodology has been developed (Sivakumar et al. 2007) for the reported anti-TB activity of chalcones, chalconelike compounds, flavones and flavanones using a statistical technique called genetic function approximation (GFA). The generated equations in each model were analyzed, for both the goodness of fit and predictive capability. The analysis also points out to the importance of the hydrogen, PMI-mag and HOMO bond donors. The study indicated that the reported compounds are more lipophilic in nature and hence, as expected, exhibit good activity since *M.tb.* has a high concentration of lipid layer. These theoretical models deserve to be explored further to design potent, newer compounds having better anti-TB activity.

#### **5. Conclusion**

Cinnamic acids, cinnamaldehydes and chalcones are unique as drug candidates in tuberculosis. Natural cinnamic-based substances such as ethyl- (**2**) and benzyl- (**4**) cinnamates not only have anti-TB activities, they are traditional medicines for hypertension as well. Cinnamaldehyde and Licochalcone A also have good potentials as anti-TB agents. Sharing a common α,β-unsaturated carbonyl functionality, these molecules offer Michaelacceptor properties, particularly to the glutathione (GSH) and cystine residues. Although, mycobacteria, unlike *E.coli*, do not prefer the formation of Michael-adduct, the presence of the cinnamic moiety certainly increased the anti-TB efficacy on several occasions. Importantly, the replacement of the double bond with an isosteric cyclopropyl ring decreased the anti-TB efficiency of the triazolophthalazines. On the other hand, introduction of cinnamoyl moiety to isoniazid did not significantly alter the trend of biological activity or the mode of action. These observations indicate that the anti-TB activity depends not only on the α,β-unsaturation but also on the functionalization of the carbonyl part of the cinnamoyl derivatives. Several hydrazone derivatives of cinnamaldehyde and chalcones have notable anti-TB activities. Importantly, substituents at the benzene ring of the cinnamic acids also play a crucial role in the biological activities. Notably, Isoprenyloxy cinnamoyltriazolophthalazine derivative (**68e**) and chalcone derived pyrazoline derivative (**102d**)

Cinnamic Derivatives in Tuberculosis 357

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#### **6. Acknowledgement**

We thank "Université Paul Sabatier" for postdoctoral grant (P.D.). Thanks are due to the European Community (integrated project "New Medicines for Tuberculosis: NM4TB 018923") and CNRS (France) for financial support.

#### **7. References**


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**16** 

*Brazil* 

**Potential Use of** *I. suffruticosa* **in Treatment of** 

**Tuberculosis with Immune System Activation** 

*Mycobacterium tuberculosis* is a serious threat to humankind, with over 8 million cases of tuberculosis (TB) annually, killing almost 3 millions of people per year around the world (WHO, 2008). Moreover, side effects from first-line anti-TB drugs can cause significant morbidity, and compromise treatment regimens for TB (Yee et al., 2003). Most healthy individuals are able to control TB infection with a vigorous immune response, halting the progression of the disease, but not necessarily eradicating the microorganism (McKinney,

The bacterium resides within macrophages, allowing them to resist the antimicrobial effector mechanisms of the host (Raupach & Kaufmann 2001). Macrophages constitute one of the main phagocyte cells of the immunological system and they are the first cells involved in an immunological response. Part of their effectiveness is due to the production of nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines, as well as phagocytosis of strange particles (Allavena et al., 2008; Carlos et al., 2004; Keil, 1999). Thus, the elimination of tuberculosis bacillus is involved in the production of these effectors molecules from immune

The hydrogen peroxide, generated by macrophages in a reaction catalyzed by an NADPH oxidase, was the first identified effector molecule that mediated mycobacteriocidal effects of mononuclear phagocytes (Lopes et al., 2005; Walker & Lowrie, 1981). In spite of several studies have indicated significant *M. tuberculosis* resistance to oxidative stress *in vitro* and *in vivo,* a recent study showed that H2O2 induced the complete sterilization of the cultures of

*M. tuberculosis* by 24 h, after the exposition to 50mM of H2O2 (Volskuill et al., 2011).

**1. Introduction** 

2000).

system.

**1.1 Tuberculosis and immune system** 

Camila Bernardes de Andrade Carli1, Marcela Bassi Quilles1, Danielle Cardoso Geraldo Maia1, Clarice Q. Fujimura Leite1,

*1Departamento de Análises Clínicas e Departamento de Ciências, Unesp,* 

Wagner Vilegas2 and Iracilda Z. Carlos1

*R. Expedicionários do Brasil 1601, Araraquara, SP, 2Departamento de Ciências Biológicas, Unesp, Rodovia Araraquara-Jaú, Araraquara, SP, 3Departamento de Química Orgânica, Unesp, R. Prof. Francisco Degni, Araraquara, SP,* 

