Natural Cinnamic Acids, Synthetic Derivatives and Hybrids with Antimicrobial Activity

Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC) of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.


Introduction
Cinnamic acids are a group of aromatic carboxylic acids (C6-C3) appearing naturally in the plant kingdom. They are formed in the biochemical route that yields lignin, the polymeric material that provides mechanical support to the plant cell wall [1]. Cinnamic acids occur in all green plants [2], OPEN ACCESS although in minute quantities covalently bound to cell walls [3], but also in the reproductive organs of flowering plants [4]. Cinnamic acids are formed in the biosynthetic pathway leading to phenyl-propanoids, coumarins, lignans, isoflavonoids, flavonoids, stilbenes, aurones, anthocyanins, spermidines, and tannins [5]. These secondary metabolites play key physiological roles in plant growth, development, reproduction and disease resistance [6,7]. The first step of this pathway is catalyzed by the phenylalanine ammonia lyase (PAL), a widely distributed phenylpropanoid enzyme present in green plants, algae, fungi, and even in some prokaryotes [8]. This enzyme deaminates L-phenylalanine to yield (E)-cinnamic acid, which undergoes other enzymatic transformations, yielding a diversity of related products [5].
The term "cinnamic" derives from the spice cinnamon (Cinnamomum zeilanicum) which has been used since antiquity as a flavoring agent and for its stimulant, carminative, antiseptic and insecticide properties [9]. The bark of several species of Cinnamomum contain considerable amounts of (E)-cinnamaldehyde, a volatile aldehyde responsible for the pungent, sweet and hot flavor of cinnamon [10,11]. Cinnamaldehyde and the essential oils of the species of Cinnamomum have antimicrobial activity both against bacteria and fungi [12,13]. Cinnamic acids are also readily available from coffee beans, tea, mate, cocoa, apples and pears, berries, citrus, grape, brassicas vegetables, spinach, beetroot, artichoke, potato, tomato, celery, faba beans, and cereals [14]. Cinnamic acids often appear as ester conjugates with quinic acid, known as the chlorogenic acids, but they can also form esters with other acids, sugars or lipids, or form amides with aromatic and aliphatic amines.
In the last ten years, the interest of researchers on the cinnamic acid moiety has notably increased. The number of published reports having the word "cinnamic" in the title, has almost doubled, from 341 in the years 1993-2003 to 633 in the period 2004-2014 according to the Scopus database (until mid-November 2014). If both "cinnamic" and "antimicrobial" keywords are used, the number of published articles increased from 1 in the period 1993-2003, to 7 in the period 2004-2014. There is no doubt that the cinnamic acids currently attracts the attention of chemists from different perspectives. Ozagrel (Figure 1), a thromboxane A2 synthase inhibitor, is in fact an imidazole para-substituted cinnamic acid that is employed therapeutically for treating acute ischemic stroke [15]. Cinromide ( Figure 1) is an antiepileptic experimental drug studied in clinical trials during the 80 decade with a favorable profile to suppress generalized convulsions, but however displayed considerable toxicity [16]. Piplartine ( Figure 1) is another cinnamic-related molecule showing an attractive biological horizon [17]. This cinnamic amide was first-time isolated from the roots of Piper tuberculatum [18], and later proved to be a promising anti-cancer scaffold [19,20].
Several reviews and studies have appeared in the literature focusing on a particular medicinal application of cinnamic-related molecules, for example on anticancer [21], antituberculosis [22], antimalarial [23], antifungal [24], antimicrobial [25], antiatherogenic [26] and antioxidant [25] activities. In addition a number of reviews directed towards the synthetic methods used to prepare cinnamic acids and related molecules have appeared in the literature [27][28][29]. Cinnamic acids have also been used by medicinal chemists to alter the potency, permeability, solubility or other parameters of a selected drug or pharmacophore. Infectious diseases caused by bacteria, fungi and viruses are still a prominent global health problem, particularly in developing, low-income countries [30,31]. Every year in the whole planet, around 4 million persons die from acute respiratory infections, around 3 million individuals die from enteric infections, around 1.8 persons die from human immunodeficiency virus (HIV), around 1.3 million die from tuberculosis (TB), and 0.7 million die from malaria [32][33][34]. Other thousand people deeply suffer the consequences of being infected by neglected tropical pathogens Schistosoma mansoni, Onchocerca volvulus, Trypanosoma spp., Leishmania spp., Mycobacterium ulcerans, Mycobacterium leprae, Wucheria spp., and others. Although some of these diseases are caused by parasites, many are caused by bacteria and fungi. Anti-bacterial and anti-fungal drugs, which are broadly known as antimicrobials, started to be used in chemotherapy since the 1940 decade [35]. New antimicrobial classes were discovered in the 1940-1970 period, and were successfully introduced to clinical practice. However as soon as the new drugs were employed, the first drug-resistant strains started to appear [36,37]. In addition the widespread use of antibiotics for animal feed and human consumption during the last forty years, has fostered the emergence of resistance in several pathogenic organisms. Infection with drug-resistant strains is typically associated with longer treatment times, higher toxicity and higher costs. Nowadays some infections are extremely difficult to treat such as extensively drug-resistant tuberculosis (XDR-TB) [38], community-associated methicillin-resistant Staphylococcus aureus (MRSA) [39] and pan-resistant Klebsiella and Escherichia coli strains [40], and they pose an enormous challenge to clinicians. The presence of these "superbugs" call for drugs with novel mechanisms of action [41]. The cinnamic skeleton is considered an interesting scaffold for the development of novel antimicrobials, however little is known about its antimicrobial mechanism of action. A recent report proposed that cinnamic acids caused fungal growth inhibition by interacting with benzoate 4-hydroxylase, an enzyme responsible for aromatic detoxification [42]. However this enzyme occurs in fungi but not in prokaryotes, and because the cinnamic acids have proven anti-bacterialeffects [43][44][45][46], other targets may be implicated in their biological effects.
This review intends to bring the attention of scientists on the antimicrobial potential of the cinnamic acids. The focus is brought to the chemical entities containing the cinnamic skeleton, displaying fungal or bacterial growth inhibition. Emphasis is placed on whole-cell inhibitory potency, structure-activity relationships and mechanism of action studies. The review is organized in different sections according to the functional groups decorating the C6-C3 cinnamic skeleton. The first section deals with natural and synthetic cinnamic acids, the second section is concerned with cinnamic esters and amides, the third section deals with cinnamic aldehydes and alcohols, and finally the fourth section covers the hybrid covalently-bound molecules between a cinnamic acid and any other biologically-relevant molecule.

Natural and Synthetic Cinnamic Acids
Honey and propolis are both bee (Apis mellifera) products made from the nectar of the flowers, and they have long been used for their antimicrobial properties [47,48]. We should recall that a report from 1978, postulated the presence of cinnamic amides in the reproductive organs of flowers [4]. Propolis and honey contain hundreds of different organic compounds, and the cinnamic acids and their esters are typically present in these natural bees products [49][50][51][52]. Cinnamic acids with varied substitution on the aryl ring, and their esters have been identified in Iranian propolis showing minimum inhibitory concentration (MIC) values between 125 and 500 mg/L against bacteria and fungi [52]. Other studies have confirmed the antimicrobial potential of propolis [53,54]. Although secondary metabolites such as flavonoids, sesquiterpenoids present in propolis may have antimicrobial activity, cinnamic acids are likely to contribute to the observed effect.
The minimum inhibitory concentration (MIC) values for the natural cinnamic acids against different bacteria, as determined by different researchers using different methods, are shown in Table 1. It was surprising to find huge differences in the MIC values for the same compounds against the same species as reported by different authors. The existence of controversial results was already noted by Wen et al., in the seminal work of the antilisterial effect of natural phenolic acids [46]. The differences may be attributed to the diversity of experimental methods for MIC determination, often measuring distinct end-points, using different inoculum sizes, different culture media, and using particular strains with varying susceptibilities [55]. Although these discrepancies obscure the antimicrobial potential of the cinnamic acid class, there is clear tendency of the molecules to inhibit the growth of a wide variety of microorganisms by molecular mechanisms that are still unknown.
Cinnamic acid (1, Figure 2) showed a weak antibacterial effect against most of Gram-negative and Gram-positive species of bacteria, with MIC values higher than 5.0 mM [12,46,[56][57][58] (Table 1). The same level of potency was observed against the fish pathogens Aeromonas hydrophila, Aeromonas salmonicida and Edwardiella tarda with MIC values between 5.6 and 7.7 mM [59]. However cinnamic acid was found to be much more active against the tuberculosis-causing bacteria, Mycobacterium tuberculosis H37Rv, with an MIC values of 270-675 µM using the SPOTi and the radiometric Bactec assays [60][61][62]. The free carboxylic acid and the presence of the α,β-unsaturation were both required for the anti-TB activity [60]. Rastogi et al., reported an MIC value of 675 µM against the H37Rv strain and varying values between 337 µM and 1.4 mM for multiple drug-resistant (MDR) clinical M. tuberculosis isolates [62]. The study found that 1 enhanced synergistically the effect of anti-TB drugs such as amikacin, ofloxacin and clofazimine. In addition its geometric isomer, cis-cinnamic acid (2) (Figure 2), was approximately 120 times more active than the trans isomer, with minimum bactericidal concentrations (MBC) values of 16.9 µM for 2, compared to 2.0 mM for 1, against an MDR M. tuberculosis strain [63]. The specific anti-TB effect of cinnamic acid may explain the traditional use of storax (Liquidambar orientalis) and cinnamon for treating TB in the 19 th century [64]. Cinnamic acid also demonstrated anti-fungal activity with MIC values of 1.7 mM against Aspergillus terreus and Aspergillus flavus, being more active against Aspergillus niger with an MIC value of 844 µM [65]. Against Candida albicans, an MIC value of 405 µM has been found [66], which is comparable to the potency against M. tuberculosis. The widely distributed natural phenol 4-hydroxycinnamic acid (3, Figure 3), also known as 4-coumaric acid, has been found to be comparatively more potent bacterial growth inhibitor compared to cinnamic acid (1) ( Table 1). However the reported MIC values of 4-coumaric acid vary to great extent from one species or strain to another. The MIC values against some Gram-negative bacteria such as Shigella disenteriae 51302 was low (MIC = 61 µM) [67], but however against Neisseria gonorrhoeae or Listeria monocytogenes or E. coli, the MIC was high (MIC > 6.0 mM) [46,58]. The same was true against Gram-positive species, with some studies reporting low MIC values for strains of Staphylococcus aureus and Bacillus subtilis whereas other reports have published higher MIC values [67][68][69][70]. A study showed that 3 disrupted the outer membrane of the Gram-negative bacteria S. disenteriae increasing the permeabilization, and in addition, the compound interacted with DNA and thus it may inhibit essential biochemical processes related to nucleic acids [67]. 4-Coumaric acid (3) completely inhibited M. tuberculosis H37Rv growth at 244 µM concentration [60], being therefore slightly more active than cinnamic acid. The compound had weak inhibition of lactic acid bacteria with MIC values of 6.09 mM [71,72] but was however relatively more active against the economically important phytopathogenic fungi Fusarium oxysporum and Fusarium verticillioides, with MIC values of 3.5 mM and 2.2 mM respectively [73]. Against Aspergillus spp MIC values have been reported between 1.5 mM and 760 µM [65].
The other abundant cinnamic acids in nature are caffeic acid (6), ferulic acid (7) and sinapic acid (8) (Figure 3), which have been studied for their antimicrobial activities [58,68,73]. A similar pattern was observed for these three natural cinnamic acids, showing a weak growth inhibition against Gram-negative bacteria compared to Gram-positive bacteria and fungi ( Table 1). The pH of the media has been reported to exert influence on growth inhibition, with lower pH values increasing the activity of the acids [76] by probably favoring a greater proportion of un-dissociated acid. The lowest MIC values for caffeic acid (6) were found against some strains of S. aureus and Streptococcus pyogenes 10535 (MIC = 694 µM) [69,77]. Caffeic acid showed significant growth inhibition of planktonic C. albicans with MIC between 694 and 710 µM [77,78]. Other species of bacteria and fungi were less susceptible to caffeic acid. Ferulic acid (7) demonstrated significant antibacterial activity against S. aureus 209 and Streptococcus pyogenes 10535 with MIC values of 644 µM [77]. Pseudomonas aeruginosa ATCC 10145 and E. coli CECT 434 were also susceptible to ferulic acid (MIC = 515 µM) [79]. In addition the strain A. niger ATCC 11394 showed a low MIC value (MIC = 322 µM) [65], however the MIC value against another strain of A. niger was found to be higher than 10 mM [80]. Interestingly A. flavus UBA 294 was the microorganism with the lowest MIC value for ferulic acid (MIC = 161 µM) [65]. A similar pattern was noticed for C. albicans, one strain being reported as susceptible with an MIC value of 659 µM [81], whereas another report showed an MIC value higher than 10 mM [80]. Sinapic acid (8), which is the most substituted of the common naturally-occurring cinnamic acids, showed significant activity against S. aureus 209 and Streptococcus pyogenes 10535 (MIC = 558 µM) [77]. The acid also demonstrated anti-Campylobacter activity with MIC values ranging from 696 µM to 1.40 mM [82]. Sinapic acid was fairly active against L. monocytogenes ATCC 7644 with an MIC value of 900 µM [83]. Sinapic acid (8) was completely inactive at a concentration of 4.46 mM against the phytopathogenic fungi F. oxysporum, A. flavus, Penicillium brevicompactum and others [73], in contrast with caffeic and ferulic acid.
Some cinnamic acids with a particular substitution pattern on the aryl ring have been prepared and examined for their antimicrobial activity. The compound 4-methoxycinnamic acid (9) was isolated from the Argentinian medicinal plant Baccharis grisebachii and its antimicrobial activity was evaluated [84]. This acid showed a potent antibacterial and antifungal effect with MIC values ranging between 50.4 and 449 µM (Table 1) [44,84]. Interestingly, the acid (9) showed higher growth inhibition against fungal species compared to bacteria, and Gram-negative and Gram-positive bacteria were equally inhibited by the compound. The acid 3,4-methylenedioxycinnamic acid (10) has been reported to inhibit Mycobacterium tuberculosis H37Rv with one report displaying an MIC value of 312 µM [60] and the other an MIC value higher than 520 µM [85]. The effect of the position of the nitro group on antimicrobial activity suggest that 4-nitrocinnamic acid (11) is more active than 3-nitrocinnamic acid (12), however the comparison data results from two different studies [44,86]. The seminal report from 1940, found that none of the positional isomers of nitrocinnamic acid inhibited S. aureus or E. coli at the highest dilution tested [87]. A noteworthy MIC value was found for 12 against the fungal species A. niger and C. albicans (MIC = 43.5 µM) [44]. Although the nitro groups can be readily reduced to amino groups, only 4-aminocinnamic (13) acid has been evaluated for its antimicrobial properties. This acid showed inhibitory activity of B. subtilis and E. coli with respective MIC values of 602 and 708 µM [86]. No information on the antimicrobial properties of the positional isomers of 4-aminocinnamic acid could be found. Although most of the halogen derivatives of cinnamic acid have been prepared [88,89], no information about their antimicrobial profile of activity was found in literature, except for 4-chlorocinnamic acid (14). This acid showed MIC values of 708 µM against both E. coli and B. subtilis [86]. Zosteric acid (15) which is naturally present in the eelgrass Zostera marina, has been found to display powerful antifouling properties by preventing bacterial biofilm formation on the surface of water-submerged objects [90]. Zosteric acid did not show any growth inhibitory activity against M. tuberculosis H37Rv [60], however the compound inhibited biofilm formation of C. albicans at 41 µM [91], but no MIC values were found in the literature search.  (19) against the same bacterial species were found to be respectively 69, 172 and 258 µM [60]. If we compare the anti-TB activity of the coumaric acids with the O-prenylcoumaric acids, it is clear that O-prenylation increases the activity for the 4-and 3-isomers, whereas for the 2-isomer, O-prenylation reduces the anti-TB activity. The C-prenylated coumaric acid drupanin (20), also known as 3-prenyl-4-coumaric acid, showed fungal growth inhibition particularly against dermatophytes species such as Epidermophyton floccosum C114, Microsporum gypseum C115, Microsporum canis C112, Trichophyton mentagrophytes ATCC 9972 and Trichophyton rubrum C113 with respective MIC values of 215, 431, 431, 431 and 431 µM [92]. Against the bacterial species, drupanin was weakly active with MIC values higher than 1.1 mM. The O-acetyl derivative of drupanin, 4-acetyl-3-prenyl-4-coumaric acid (21) showed a similar pattern of activity, being able to inhibit the dermatophyte E. floccosum C114 with an MIC value of 364 µM [92]. It was therefore slightly less active than the non-acetylated compound.    The compound 3,5-diprenyl-4-coumaric acid (22) was comparatively more active growth inhibitor of E. floccosum C114 achieving an MIC values of 166 µM [92]. Its O-acetyl derivative (23) was again slightly less active than the non-acetylated compound ( Table 1), suggesting that the free phenolic OH is essential for potent activity of this class of molecules. The 4-O-acetyl derivative of ferulic acid, namely 4-O-acetylferulic acid (24), prepared by synthesis, inhibited the growth of C. albicans, Candida krusei and Enterococcus faecalis at 540 µM, but was less active against S. aureus, E. coli and Klebsiella pneumoniae [81]. Its activity was found to be comparatively similar to the antimicrobial effect of ferulic acid.

Esters
The chlorogenic acids are a family of natural esters of hydroxycinnamic acids (coumaric, caffeic, ferulic and sinapic acids) with (−)-quinic acid [14]. The most common chlorogenic acid is 5-O-caffeoylquinic acid (25, Figure 4), which is abundant in coffee, black tea and mate but is also present in apples, pears and berries [95]. This chlorogenic acid isolated from artichoke, displayed MIC values of 564 µM against B. subtilis, S. aureus, E. coli, S. typhimurium, P. aeruginosa and S. cerevisiae, and lower MIC values of 282 µM against Micrococcus luteus, Agrobacterium tumefaciens, Aspergillus niger, Penicillium oxalicum and Mucor mucedo [96]. It was even more active against the fungi Candida albicans, Candida lusitaniae, Saccharomyces carlsbergencis and Cladosporium cucumerinum with an MIC value of 141 µM [96]. MIC values for the natural cinnamic esters are summarized in Table 2. However the effect of (25), isolated from roasted coffee beans, against S. aureus and Streptococcus mutans was much less marked in the study of Daglia [101], and was therefore comparatively more active than the parent chlorogenic acid. Transmission electron microscopy, membrane potential and nucleotide leakage studies on Shigella disenteriae led to the conclusion that the chlorogenic acid disrupted cell wall permeability and then depolarized the bacterial cell wall membrane causing cytoplasmic leakage [100].
4-O-Caffeoylquinic acid (26) ( Figure 4 and Table 2) was found to be slightly less active than the 5-O-isomer with MIC values of 423 µM against both S. aureus and E. coli, 564 µM against both fungi C. albicans and A. niger, and 705 µM against S. enterica and S. cerevisiae [98]. In contrast with 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid (27) displayed more pronounced growth inhibitory activity against bacteria than fungi (Table 2), achieving MIC values of 282 µM against E. coli and S. aureus [98]. The literature reports of the antimicrobial activity of the 3-O-and 4-O-isomers are scarce, in contrast with the 5-O-isomer. The fact that the esters 25, 26 and 27 have a different antimicrobial profile suggests that they have antimicrobial activity on their own, and the hydrolysis products caffeic and quinic acids, which could be released in the same amount, are not responsible for the antimicrobial activity of the three esters.  Among the chlorogenic acids formed by two caffeic acids residues, 1,3-di-O-caffeoylquinic acid (28), 3,5-di-O-caffeoylquinic acid (29) and 4,5-di-O-caffeoylquinic acid (30), the presence of caffeic residue in the position 3 of quinic acid increased the antifungal spectrum of activity, with 28 achieving MIC values below 200 µM against almost all fungi tested (Table 2), whereas the esterification in the position 5 of quinic acid increased the antibacterial potency of the acid, displaying MIC values of 194 µM against E. coli and S. aureus for 30 and even inhibiting completely the growth of Micrococcus luteus at 97 µM [96]. However discrepant MIC have also been reported. A report from 2011, published MIC values higher than 248 µM against S. aureus, B. cereus and higher than 496 µM against E. coli and C. albicans for 28, 29 and 30 [99]. Moreover the study found an impressive synergism (FICI < 0.002) between 30 and several fluoroquinolone antibiotics against S. aureus. Four caffeic acid glycosides isolated from Paulownia tomentosa displayed antibacterial activity [102]. Campneoside I (32) demonstrated the highest activity against Gram-positive bacteria achieving MIC values around 200 µM against different species of S. aureus, while both acteoside (31) and campneoside II (33) showed less antistaphylococcal activity [102].
The caffeic acid ester 34 ( Figure 4) isolated from Zuccagnia punctata, showed antifungal activity against the phytopathogenic fungi Phomopsis longicolla with an MIC value of only 19 µM, being less active against other fungi [103]. Another caffeic acid ester known as rosmarinic acid (35), and its methyl ester 36 showed growth inhibition of several species of bacteria with MIC values ranging between 801 µM and 6.94 mM [104]. In the study, the methyl ester 36 was slightly more active than the free acid 35. However both rosmarinic acid (35) and its methyl ester 36 isolated from Rabdosia serra, were reported to have significantly higher MIC values against Gram-positive and Gram-negative bacteria [105], and surprisingly the methyl ester was found to be completely inactive with MIC values higher than 3.4 mM, whereas rosmarinic acid showed some activity [105] (Table 2). Moreover the study of Gohari et al., showed that Candida albicans was more susceptible to rosmarinic acid with an MIC value of 694 µM, in comparison with S. aureus, E. coli and Aspergillus niger [106]. Caffeic acid phenethyl ester (37) is a biologically active component of propolis showing interesting anticancer activity [107]. This ester demonstrated antibacterial activity against S. aureus, E. faecalis and L. monocytogenes with MIC values ranging between 100 and 400 µM, but did not show growth inhibition against P. aeruginosa and E. coli up to a concentration of 800 µM [108]. Trilepisiumic acid (38) is an ester of caffeic acid and protocatechuic acid, isolated from Trilepisium madagascariense, that shows antifungal activity against Candida albicans ATCC 9002 (MIC = 202 µM) while being less active against other fungi and bacteria [109]. Two cinnamic esters were isolated from the root of Ehretia longiflora, and identified as ehretiolide (39) and arachidyl ferulate [110]. Ehretiolide (39) showed growth inhibitory activity against M. tuberculosis H37Rv (MIC = 41 µM), which is impressive for an unmodified natural product.

Amides
The simplest cinnamic amide is cinnamide (78) ( Figure 5 and Table 3) which displayed growth inhibition against both fungi A. niger and C. albicans at a concentration of 60.8 µM, while being less active against bacteria, showing an MIC value of 252 µM against B. subtilis, E. coli and S. aureus [44]. All the cinnamoyl amides 79-94 were found to have a more potent effect against fungi compared to bacteria, except cinnamoyl dopamine (94) which showed little antimicrobial activity (MIC = 1.76 mM) [77]. Among the screened cinnamoyl amides, the compound with the highest antifungal activity was identified to be cinnamoyl N,N-diethylamide (84)     The 4-coumaroyl amides 95-115 were tested for antimicrobial activity, and the MIC results suggest that they are comparatively more active inhibitors of microbial growth than the cinnamoyl amides ( Table 3). The 4-coumaroyl amides do not show a higher specificity for fungi compared to bacteria as was observed for the cinnamoyl amides. The MIC values of the 4-coumaroyl amides 95-115 ranged between 929 nM and 3.6 mM against bacteria, and between 6.3 µM and 18 mM against fungal species [77,111]. The 4-coumaroyl amide displaying the lowest MIC value against bacteria, was found to be 4-coumaroyl 2'-chloro-4'-nitrophenylamine (111) achieving an MIC value of 929 nM against B. subtilis MTCC 2063 [111], while the lowest MIC value against fungi was 6.3 µM attained by 4-coumaroyl 2'-nitrophenylamine (103) against C. albicans MTCC 227 [111]. Most of the 4-coumaroyl amides displayed higher activity against B. subtilis and S. aureus bacteria compared to E. coli ( Table 3).
The caffeoyl amides 116-119 ( Figure 5) showed moderate antimicrobial activity (Table 3) with MIC values ranging between 194 µM and 1.76 mM [77]. The lowest MIC value (194 µM) was reported for caffeoyl tryptamine (119) against S. pyogenes 10535, however the MIC values for the same amide against other bacteria were comparatively higher, being 1.55 mM against B. subtilis 1A95, C. albicans 62 and L. monocytogenes C12 [77]. The amides caffeoyl dopamine (117) and caffeoyl tyramine (118) showed growth inhibition against Gram-positive, Gram-negative and fungal species with MIC values between 396 and 793 µM, and 418 and 836 µM respectively, whereas the other caffeoyl amides displayed MIC values higher than 1.0 mM against at least one microorganism ( Table 3). The three evaluated feruloyl amides 120-122 showed a very similar pattern of antimicrobial activity [77]. The feruloyl amides showed to be growth inhibitors of S. aureus 209 with MIC values between 190 and 372 µM. They were equally active against S. pyogenes but much less against B. subtilis (MIC around 743-798 µM), and C. albicans and L. monocytogenes. Interestingly all the sinapoyl amides 123-126 showed selective growth inhibition of Gram-positive cocci bacteria (MIC between 171 and 696 µM), while being much less effective inhibitors of B. subtilis, L. monocytogenes or C. albicans (MIC between 1.36 and 1.53 mM) [77].
The Piper amides, which are one of the major phytochemicals present in the medicinally important Piper species displayed a broad variety of biological activities [113]. The carboxylic acid residues of some Piper amides are in fact substituted cinnamic acids.
Piplartine (Figure 1), originally isolated from the roots of Piper tuberculatum [18], demonstrated antibacterial activity against K. pneumoniae, P. aeruginosa and S. aureus, however no MIC values were reported [114]. Among the amides isolated from the seeds of Piper tuberculatum, piplartine showed antifungal activity against Cladosporium sphaerospermun but no MIC value was determined [115,116]. The amide 3',4',5'-trimethoxycinnamoyl pyrrolidine (127), which is related to piplartine, was isolated from Piper sarmentosum, and showed MIC values higher than 600 µM against M. tuberculosis H37Ra [117,118]. The toussaintines are amides of cinnamic acid with indolidinones or benzofuranones, obtained from the African plant Toussaintia orientalis [119]. The toussaintine B (129) inhibited both S. aureus and E. coli with a low MIC value of 67 µM, while toussaintine A (128) inhibited E. coli at 34 µM but did not inhibit S. aureus, and toussaintine D (131) inhibited S. aureus at 17 µM but did not inhibit E. coli [119]. Reports of natural cinnamic amides displaying antimicrobial activity were very scarce in the literature.

Natural and Synthetic Cinnamic Aldehydes and Alcohols
Cinnamaldehyde (132) (Figure 6, Table 4) which is abundantly obtained from the essential oils of cinnamon [12,120], has demonstrated a significant antimicrobial activity particularly against Gram-negative bacteria. The aldehyde was found to be very active against Helicobacter pylori and E. coli with MIC values of 15 and 7.6 µM respectively [121]. Similar MIC values of 10.6 µM and < 1.5 µM have been reported against P. aeruginosa and E. coli respectively [122]. However controversial results have appeared for this compound, as other reports published higher MIC values against E. coli [12,[123][124][125]. Against the Clostridium difficile bacteria, the aldehyde showed complete growth inhibition at a concentration of 605 µM [13]. A similar MIC value was observed against Legionella pneumophila, the causative bacteria of Legionnaire's disease [126]. Cinnamaldehyde has also found to be more active against S. aureus than Gram-negative bacteria [124], and interestingly the MIC values against a susceptible S. aureus and a methicillin-resistant S. aureus (MRSA) were exactly the same. The antibiotic clindamycin actually was found to be synergistic with cinnamaldehyde, with a FICI value of 0.3125 [124]. In addition, cinnamaldehyde showed interaction with the FtsZ cell division protein using isothermal titration calorimetry, and inhibited the formation of FtsZ assembly in a dose-dependent manner, which phenotypically translated into elongated cells [124]. A report from 2003, showed that cinnamic aldehyde prevented Bacillus cereus daughter cells separation during division, forming filamentous cells [127], a result correlating with inhibition of FtsZ. Against S. aureus clinical isolates the reported MIC values were 5.0 and 10 mM, however cinnamaldehyde was found to inhibit biofilm formation at concentration five times higher than the MIC [128]. Cinnamaldehyde (25) showed an MIC value of 358 µM against Fusarium verticillioides, the phytopathogenic fungi infecting maize that can also produce mycotoxins during the storage of grains [129]. Treatment of F. verticillioides with the aldehyde produced morphological alterations of the hyphae and the cell wall, which resulted in cytoplasmic leakage. Cinnamaldehyde showed weak antifungal activity against C. albicans and Candida tropicalis with MIC values of 3.0 and 3.8 mM respectively [130], however lower MIC values have been reported [131,132]. Against wood decay fungi, Laetiporus betulina and Laetiporus sulphureus the potency was comparable, with MIC values around 750 µM [133,134].
From the medicinal plant Piper taiwanense, caffeic aldehyde (133) (Figure 6) was isolated and found to have significant antitubercular properties (MICH37Rv = 154 µM) [135]. The other natural brothers of cinnamaldehyde, coniferaldehyde (134) and sinapaldehyde (135) displayed moderate anticandidal activity [136]. More than 60 strains and clinical Candida isolates were evaluated for their susceptibility to the three aldehydes. Sinapaldehyde was found to exert the most potent effect with a MIC values between 480 and 960 µM, while the MIC value of coniferaldehyde and cinnamaldehyde varied between 914 µM and 1.83 mM, and between 1.14 and 3.78 mM respectively [136]. Phenotypic characterization of C. albicans treated with sub-inhibitory concentrations of the aldehydes showed alterations in cell morphology and the cell wall, as well as damage to the plasma membrane resulting in cell lysis and cytoplasmic leakage. The study suggested inhibition of membrane ATPases, by examination of H + efflux rates in presence of the aldehydes and known ATPase inhibitors [136]. Both coniferaldehyde (134) and sinapaldehyde (135) showed growth inhibitory properties against three species of Streptococcus with MIC ranging from 150 µM to 1.4 mM, being 135 more active against S. pyogenes and S. mutans, while 134 was more active against S. mitis [137].
Interestingly the most active antifungal compound present in cinnamon was identified to be 2-methoxycinnamaldehyde (136), achieving MIC values as low as 19 µM against Microsporum canis [138], a fungi causing ringworms in pets and tinea capitis in humans. This aldehyde was also active against other fungi including C. albicans, with an MIC value of 308 µM, but was however less active against A. niger and different species of bacteria [138]. The positional isomer 4-methoxycinnamaldehyde (137) was less active with MIC values of 770 µM against C. albicans MTCC 3017, Issatchenkia orientalis MTCC 231, P. aeruginosa MTCC 424 and Trichophyton rubrum MTCC 296, and higher values (MIC > 3 mM) against other microorganisms [139]. Clearly, as observed for the cinnamic acids, the substitution on position 2 of the cinnamic skeleton, confer an increase in the antimicrobial effect. The presence of another methoxy groups in position 3, did not provide a substantial effect, as 3,4-dimethoxycinnamaldehyde (138) showed MIC values higher than 2.5 mM for all the evaluated microorganisms (Table 4), except against T. rubrum MTCC 296 (MIC = 160 µM) [139]. The aldehyde having three methoxyl groups, 3,4,6-trimethoxycinnamaldehyde (139), showed a similar profile of activity, being a weak growth inhibitor of species of Aspergillus, Candida, Issatchenkia and Micrococcus (MIC > 1.4 mM) but being active against the dermatophyte T. rubrum MTCC 296 with an MIC value of 280 µM [139]. The compound 3,4-methylenedioxycinnamaldehyde (140) showed to be a potent inhibitor of T. rubrum fungi (MIC = 40 µM), with moderate activity against A. sydowii, C. albicans, I. orientalis and P. aeruginosa with an MIC value of 350 µM [139]. The aldehyde was less active against other bacteria and fungi. The replacement of a methoxy group in position 4 of 138 for an ethoxy group, as in 3-methoxy-4-ethoxycinnamaldehyde (141), slightly decreased the MIC values for most of the compounds, however the specificity against T. rubrum and I. orientalis decreased substantially. Introduction of a bromide to 138, as in 2-bromo-3,4-dimethoxycinnamaldehyde (142) decreased the antifungal activity against T. rubrum, increasing the MIC value to 460 µM [139]. The compound N,N-dimethyl-4-aminocinnamaldehyde (143) was the only tested cinnamaldehyde having an amino group, and displayed an MIC of 180 µM against T. rubrum, but was inactive against the bacteria Enterobacter cloacae and Micrococcus luteus (MIC > 11 mM). The cinnamaldehyde having a nitro substituent 2,2-nitro-4-methylcinnamaldehyde (144) showed a similar antimicrobial profile as the other cinnamaldehydes, with a potent growth inhibitory effect against T. rubrum (MIC = 160 µM) but with low activity against several species of bacteria (MIC > 2.5 mM) [139]. Cinnamyl alcohol (145) is a component of several essential oils, particularly those of Cinnamomum species and other Lauraceae plants [140][141][142]. Cinnamyl alcohol showed little inhibitory effect against all the evaluated bacterial and fungal species (Table 4) [12,126,143]. The study of Barber et al. published in 2000, reported the antimicrobial activities of 4-coumaryl alcohol (146), coniferyl alcohol (147) and sinapyl alcohol (148) [68]. Comparatively the alcohols were found to be less active than their corresponding aldehydes, showing MIC values equal or higher than 8.0 mM against bacteria and yeasts. Cinnamyl benzoate (149) showed little activity against Gram-negative and Gram-positive bacteria (MIC > 10 mM), however it was higly active against dermatophyte fungi Trycophyton sp. and Epidermophyton floccosum IFO 9045 (MIC = 20 µM) [143]. Two acetate esters of coumaryl alcohol displayed growth inhibition of some fungi and S. aureus [144]. 4-Coumaryl acetate (150) displayed MIC values higher  [144]. The diester 4-coumaryl diacetate (151) showed higher anti-fungal activity in comparison with 150, and additionally the diacetate demonstrated significant antimycobacterial activity with an MIC value of 215 µM against Mycobacterium smegmatis mc 2 -155 [145].

Synthetic Cinnamic Hybrids
The interest in making hybrids molecules result from the idea of combining the biological properties of two active molecules to yield a third molecule (chimera or mermaid) with a stronger effect [147]. The conjugation can be used to modulate selectivity, spectrum of activity, potency, and physicochemical parameters. Theoretically the hybrid molecule once administered to biological systems could be lysed to yield in situ the two active molecules with atomic economy, or the hybrid molecule could be made sterically flexible enough to act on the two domains of the biological targets as a single piece [148]. Several cinnamic hybrids have been prepared [149][150][151][152][153] but only a few of them have been evaluated for their antimicrobial properties.
The studied cephem hybrids 152-155 ( Figure 6 and Table 5) are cephalosporin moieties linked covalently to a 2,5-dichlorocinnamic (Figure 4). These hybrids showed a potent growth inhibitory effect against Staphylococcus epidermidis A24548, Staphylococcus haemolyticus A21638 and S. pneumoniae A28272 with MIC values in the nanomolar range [154]. The cephem 155 having a cinnamic aldehyde and a 2-hydroxy-3-propylamine N-substitution on the pyridine ring, was the most active against S. epidermidis A24548 (MIC = 22 nM) and S. haemolyticus A21638 (MIC = 87 nM). The oxazolidinone hybrid (156) showed strong antibacterial activity against E. faecalis ATCC 29212 (MIC = 194 nM), Enterococcus faecium ATTC 700221 (MIC = 388 nM) and S. aureus ATCC 29213 (MIC = 194 nM) [155]. Against the methicillin-resistant S. aureus ATCC 33591, the cinnamic hybrid (156) displayed an MIC value of 1.6 µM. A peptide hybrid based on the human cysteine protease inhibitor cystatin C has been prepared and evaluated for its antimicrobial properties [156]. The peptide cystapep 1 (157), having a cinnamic acid residue, showed MIC values of 25.2 µM against S. aureus and Streptococcus pyogenes and slightly higher MIC values against other Streptococcus species. This is the only antimicrobial peptide cinnamic hybrid known so far. The rifamycin hybrid SV (T9) (158) (Figure 6) having a cinnamic acid residue linked through amide bond with the piperazine ring, is slightly more potent than rifampin (MICT9 = 106 nM vs. MICRIF = 243 nM) [157]. The molecule has demonstrated to be useful in clinical application [158]. The MIC values of the hybrid molecule were in the nanomolar range against the pathogenic mycobacteria Mycobacterium avium complex, Mycobacterium leprae and M. tuberculosis [157,159,160]. However the hybrid T9 is not active against rifampin-resistant M. tuberculosis strains.
Among the prepared 4-coumaric hybrids from the same comprehensive study, the cycloserine hybrid 165 displayed an MIC value of 950 µM [161], while the literature MIC value of cycloserine is 245 µM [161,163]. Nonetheless the study identified the triazolophtalazine cinnamic derivative 166 with a significant MIC value of 1.4 µM against the H37Rv strain and a selectivity index around 320 in comparison to THP-1 cells [161]. Guanyl hydrazone hybrids have also been prepared and examined for antimycobacterial activity [164]. Hydroxy substitution of the benzaldehyde hydrazone increased the anti-TB activity, with the hybrid 167 having a hydroxyl in position 4 displaying an MIC value of 40.5 µM against the virulent H37Rv strain [164]. The presence of 3,4-dimethoxy substitution on the benzaldehyde hydrazone, as in the hybrid 168, increased growth inhibition (MIC = 8.9 µM).
Thirty-one fenchol hybrids were prepared with different substitutions on the cinnamic ring, and four hybrids 169-172 resulted with potent activity against M. tuberculosis H37Rv [165]. The fenchol hybrids 169 and 170 having respectively a 3,4-methylenedioxy and a 2-nitro substituents (170) on the aryl ring of the cinnamic moiety, displayed an MIC value of 6.7 µM ( Table 5). The fenchol hybrid 171 with 3,4,5-trimethoxy substitution on the cinnamic acid showed higher potency (MIC = 2.4 µM), while the hybrid 172 having 4-dimethylamine substitution was the most active (MIC = 540 nM) against the H37Rv pathogenic strain [165].     In a study from 2008, three triterpenes, betulinic, oleanolic and ursolic acids, were esterified in the 3-hydroxyl position, with different cinnamic acids with the aim of generating novel molecules which could potentially inhibit the M. tuberculosis H37Rv bacteria [166]. The cinnamic acids employed for the preparation of the esters were cinnamic, 4-coumaric, caffeic and ferulic acids. The esters having the 4-coumaroyl moiety, 3-O-(4'-coumaroyl) oleanolic acid (174), 3-O-(4'-coumaroyl) ursolic acid (178) and 3-O-(4'-coumaroyl) betulinic acid (182) were among the esters with the highest anti-TB activity, achieving MIC values of 10.4 µM. The MIC values of the free triterpenoids were 109 µM for oleanolic and betulinic acids, and 27.4 µM for ursolic acid [166], and therefore conjugation with 4-coumaric acid increased the anti-TB potency. Moreover the ester with the highest activity, 3-O-feruloyl ursolic acid (180), was able to inhibit completely the growth of M. tuberculosis at a concentration of 4.95 µM. The cinnamate and caffeate esters showed moderate to little anti-TB activity, with MIC values higher than those obtained for the free triterpenoids.

Conclusions
This review summarizes the in vitro antimicrobial activity of several cinnamic-related molecules by collating the reported MICs in a comprehensive list. Because the MIC data included in this review was extracted from several studies (using different experimental methods), it is far from ideal to compare the MIC as absolute values, but rather the MICs should be used as relative numbers indicating the tendency of the compounds to inhibit certain microorganisms. This review primarily serves as a framework to quickly identify the cinnamic acids and related molecules that have been tested for their antimicrobial properties.
Among the cinnamic-related molecules with the highest antimicrobial activity, the hybrids between antibiotics and cinnamic acids, such as the rifamycin T9 (158) and the oxazolidinone 156 with MIC values in the nanomolar range, were the champions. However the activity of these hybrids is mostly due to the potent effect of the antibiotic component. It is unknown whether the conjugates actually hydrolyze in vivo to yield two active molecules with potential synergism, or it is the whole molecule responsible for the observed biological effect. Among the non-hybrid cinnamic-related molecules with the lowest MIC values 4-methoxycinnamic acid (9), 3-nitrocinnamic acid (11), all the caffeoyl quinic acids 25-30, most of the cinnamate and 4-coumarate esters 40-61, and most of the cinnamoyl and 4-coumaroyl amides 78-115 are worth mentioning. A remarkable antimicrobial activity was detected for isobutyl cinnamate (45) achieving a broad spectrum of activity against yeasts, Gram-positive and Gram-negative bacteria, with MIC values between 43 and 12 µM. Further studies need to confirm the potent antimicrobial activity observed, expanding the screening to other microorganisms and drug resistant-isolates, and finally evaluating its toxicity. This example illustrate how a simple substitution, for instance comparing isobutyl cinnamate (45) with butyl cinnamate (44), can have a significant impact on the biological properties of the molecules (4-fold MIC change against some microorganisms).
There is no doubt that the cinnamic acids, their derivatives and hybrid molecules display marked antimicrobial effects. Some microorganisms are more sensitive to a chemical class than others. For instance, fungal organisms are generally more susceptible to the cinnamic aldehydes, while the cinnamic acids, esters and amides tend to affect more importantly the bacteria. A noteworthy effect was observed for the cinnamic molecules against Mycobacterium tuberculosis. The growth of the TB-causing bacteria was repeatedly inhibited by micromolar concentrations of molecules containing the cinnamic acid moiety. However very little is known about the mechanism of action of cinnamic acid, and the essential structural features required for anti-TB activity. Detailed molecular studies of the biological targets of the cinnamic acids may help to design high-affinity cinnamic-based ligands which may be important for developing future therapeutic alternatives to the growing problem of drug-resistant microbial pathogens.