Antimycobacterial plant terpenoids, CL Cantrell, SG Franzblau, NH Fischer

Tags: tuberculosis, antimycobacterial, Charles L. Cantrell, Sigma-Aldrich, Georg Thieme Verlag Stuttgart, terpenoids, synthetic analog, Mycobacterium tuberculosis, ZUrich Editorial Advisory Board Yoshinori Asakawa, development, MUnster Co-Editors Wolfgang Barz, rifampin, New York, Tallahassee Manfred Hesse, Louisiana Gerhard Franz, Gif-sur-Yvette Yarra E. Tyler, Stuttgart Thieme New York, Adolf Nahrstedt, Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School, oleanolic acid, Salvia, Biological activity, Acaena pinnatifida, betulinic acid, fusidic acid, Sigma-Aldrich Co., Tokushima Gatz Harnischfeger, Scott G. Franzblau2, ursolic acid, Otto Sticher, DUsseldorf Nikolaus H. Fischer, Franzblau SG, Journal of Natural Products, Fischer NH, Cantrell CL, Antimicrobial Agents and Chemotherapy, European Respiratory Journal, analogs, initial activity, structural analogs, Mohamed S. Rajab, Planta Medica, Miyazaki E, M. tuberculosis, Institute for Tuberculosis Research, antitubercular agents, West Lafayette Pieter A. van Zwieten, Agricultural Research Service, Chase S. Activities, Rios T. Hydroxy-bis-dihydroencelin, Cantrell, Phytochemistry, National Center for Agricultural Utilization Research
Content: Editor Adolf Nahrstedt. MUnster Co-Editors Wolfgang Barz. MUnster Rudolf Bauer. DUsseldorf Nikolaus H. Fischer. Baton Rouge. Louisiana Gerhard Franz, Regensburg Walter E. MUller. Frankfurt/M. Otto Sticher. ZUrich Editorial Advisory Board Yoshinori Asakawa, Tokushima Gatz Harnischfeger, Salzgitter Werner Herz, Tallahassee Manfred Hesse. ZUrich Kurt Hostettmann. Lausanne Pierre Potier. Gif-sur-Yvette Yarra E. Tyler. West Lafayette Pieter A. van Zwieten. Amsterdam
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Antimycobacterial Plant Terpenoids Charles L. CantreIl 1.*, Scott G. Franzblau2, Nikolaus H. Fischer3 1 U.S. Department of Agriculture, Agricultural Research Service. National Center for Agricultural Utilization Research, Peoria. Illinois, USA 2 Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA 3 Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, Oxford, Mississippi, USA Received: June 13. 2001; Accepted: July 10, 2001
Abstract: Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis, is the leading killer among all infectious diseases worldwide and is responsible for more than two million deaths annually. For over thirty years no antitubercular agents with new mechanisms of action have been developed. The recent increase in the number of multi-drug resistant clinical isolates of M. tuberculosis has created an urgent need for the discovery and development of new antituberculosis leads. This review covers recent reports on plant-derived terpenoids that have demonstrated moderate to high activity in in vitro bioassays against M. tuberculosis. In this review, mono-, sesqui-, di- and triterpenes, and sterols, their structural analogs and semisynthetic derivatives will be discussed, with particular emphasis on the structural features essential for anti mycobacterial activity. Key words: Terpenoids, Mycobacterium tuberculosis, antituberculosis, tuberculostatic, anti mycobacterial. Introduction It is estimated that one-third of the world's population is infected with the tubercle bacillus (1). While only a smaIl percentage of infected individuals will develop clinical tuberculosis, each year there are approximately eight miIIion new ca- ses and two miIlion deaths. M. tuberculosis is thus responsible for more human mortality than any other single bacterial species. The HIV pandemic has exacerbated the problem by providing a large reservoir of highly susceptible individuals (2). A number of efficacious antitubercular agents were discovered in the late 1940's and 1950's with the last, rifampin, introduced in the 1960's (3), (4). These agents have reasonable efficacy and when used in combination should preclude the development of drug resistance. The use (or in most cases misuse) of these drugs has lead over the years to an increasing prevalence of multiple-drug resistant (MDR) strains and there is now an urgent need to develop new effective agents (5), (6). There have been a number of practical obstacles to the development of new anti-TB agents, among them a lack of econom- Planta Med 67 (2001) 685 - 694 © Georg Thieme Verlag Stuttgart· New York ISSN: 0032-0943
ic incentive due to the predominance of disease in the developing world. The very slow growth and highly contagious na- ture of M. tuberculosis have also served to discourage the drug discovery effort. Nonetheless, several drugs with interesting anti-TB activity have been identified in the past few years; three such compounds are the rifamycins. Rifabutin, with less P450 activating activity than rifampicin. has activity against a small percentage of rifampin-resistant strains (7), (8) and is active c1inicaIly (9), (10), (11), (12). Rifapentene provides a much longer serum half-life and thus holds the promise of allowing for intermittent dosing but exhibits complete cross-resistance with rifampin (13). KRM-1648 appears to be the most potent among the rifamycins and has demonstrated activity in phase II trials in tuberculosis (14); however, it also exhibits partial cross-resistance with rifampin. Fluoroquinolones such as ofloxacin and levofloxacin have demonstrated clinical activity (15), (16), (17), (18); however, the recently approved (in the U.S.A. for non-TB respiratory indications) moxifloxacin ap- pears to be the most active flouroquinolone against M. tuberculosis in vitro and in the mouse (19), (20), (21). Two members of a new class of antimicrobials, the oxazolidinones, showed modest in vivo activity against M. tuberculosis (22). Finally a nitroimidazopyran has demonstrated interesting in vitro and in vivo activity (23). While all of these compounds show potential, none have yet to demonstrate activity in clinical trials (with the exception of KRM-1648 and the older quinolones), thus there continues to be a need to identify new agents. The following review surveys the literature for plant-derived terpenoids that have demonstrated moderate to significant biological activity against M. tuberculosis. It covers active compounds of five major groups of terpenoids: monoterpenes, sesquiterpenes, diterpenes, triterpenes, as weIl as phytol and its derivatives and structural analogs. Nearly all of the compounds discussed were screened using the BAGEC 460 system (24), (25) except where indicated otherwise. Compounds that did not demonstrate a Minimum inhibitory concentration (MIC) of 64,ug!ml or lower and were unrelated to active terpenoids, have been omitted. Comparisons between chemical structures and their biological activities must be treated with caution, especially when different bioassay techniques have been used.
686 Planta Med 67 (2001)
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Table 1 Minimum inhibitory concentrations (MICs) of selected plant terpenoids against M. tuberculosis (H37Rv)
Compound Class Name
MIC tug/ml)'
Antituberculosis Drugs isoniazid rifampin streptomycin ethambutol pyrazinamide
Sigma-Aldrich Co.
Sigma-Aldrich Co.
Sigma-Aldrich Co.
Sigma-Aldrich Co.
Monoterpenes citronellol (1) nerol (2) geraniol (3)
Sigma-Aldrich Co.
Sigma-Aldrich Co.
Sigma-Aldrich Co.
Sesquiterpenes costunolide (4) 11 {JH-dihydrocostunolide (5) parthenolide (6) 11 {JH-dihydroparthenolide (7) l,10-epoxycostunolide (8) l,10-epoxydihydrocostunolide (9) l,10-epoxyparthenolide (10) l,10-epoxydihydroparthenolide (11) a-cyclocostunolide (12) santa marine (13) 11 {JH,13-dihydrosantamarine (14) {J-cyclocostunolide (15) reynosin (16) 11 {JH,13-dihydroreynosin (17) a-santonin (18) reynosin triol derivative (19) 11,13-dihydro triol of reynosin (20) santa marine triol derivative (21) dehydrocostuslactone (22) 7-hydroxydehydrocostuslactone (23) zaluzanin C (24) dehydrocostuslactone, 4a(15)-epoxide (25) dehydrocostuslactone, 10{J(14)-epoxide (26) dehydrocostuslactone, 1Oa(14)-epoxide (27) dehydrocostuslactone, 4{J(15),l Oa(14)-diepoxide (28) dehydrocostuslactone, 4a(15),l Oa(14)-diepoxide (29) dehydrocostuslactone, 4a(15),l 0{J(14)-diepoxide (30) micheliolide (31) pumilin (32) damsin (33) deacetylconfertifJorin (34) confertifJorin (35) deacetylisoconfertifiorin (36) parthenin (37) tenulin (38) peruvin (39) peruvin acetate (40) burrodin (41) aromaticin (42) alantolactone (43) isoalantolactone (44) encelin (45) l,2-dehydro-3-epi-isotelekin (46) ivalin (47) 11 aH,13-dihydroisoalantolactone (48) isoalloalantolactone (49) alloalantolactone (50) 3-oxoa!loalantolactone (51) 4a-epoxyisoalantolactone (52) 5a-epoxyalantolactone (53)
Saussurea lappa synthetic analog Magnalia grandiflora Ambrosia ortemisiifolia synthetic analog synthetic analog synthetic analog synthetic analog synthetic analog Ambrosia confertiflara Sanchus hierrensis synthetic analog Ambrosia confertiflora synthetic analog Artemisia ramosa Saussurea loppa Podachenium eminens Za/uzania trl/oba synthetic analog synthetic analog synthetic analog synthetic analog synthetic analog synthetic analog Berlandiera texano Ambrosia maritima Ambrosia confertiflora Ambrosia confertiflora synthetic analog Parthenium hysterophorus Helenium amarum Ambrosia peruviana synthetic analog Ambrosia dumosa Helenium aromaticin Inula helenium Inula helenium Montanoa speciosa Montanoa speciosa Iva imbricata Inula helenium Rudbeckia mollis Rudbeckia subtomentosa Rudbeckia subtomentosa synthetic analog synthetic analog
32 128 16 128 64 128 128 128 64 64 >128 64 64 >128 >128 >128 >128 >128 2 >128 >128 32 32 64 64 128 128 50 128 32 128 128 128 64 128 128 >128 128 16 32 32 16 32 64 >128 128 32 128 32 8
(28) (28) (28) (28) (28) (28) (28) (28) (26) (28) (28) (26) (28) (28) (26) (28) (28) (28) (30) (30) (30) (30) (30) (30) (30) (30) (30) (30) (30) (26) (41), (26) (41), (26) (26) (26) (26) (26) (26) (42) (26) (31) (31) (31), (43) (31), (43) (26) (31) (44), (31) (31) (31) (31) (31)
Antimycabacterial Plant Terpenoids Tablel cant. Compound Class Name 11,13-dihydroxyalantolactone (54) 6-epi-deacetyllaurenobiolide (55) 4,5-epoxy-6-epideacetyllaurenobiolide (56) curcuphenol (57) nerolidol (58) farnesol (59) Diterpenes sandaracopimara-8(14)-15-diene-7 a,18-diol (60) sandracopimaric acid (61) sclareol (62) 12-demethylmulticauline (63) multicaulin (64) 12-demethylmultiorthoquinone (65) multiorthoquinone (66) 12-methyl-5-dehydrohorminone (67) 12-methyl-5-dehydroacetylhorminone (68) salvipimarone (69) 9,12-cyclomulin-13-ol (70) juniperexcelsic acid (71) Triterpenes ergosterol-5,8-endoperoxide (72) ergosterol-5,8-endoperoxide acetate (73) ergosterol (74) 12j3-hydroxykulactone (75) 6f3-hydroxykulactone (76) kulonate (77) (24R)-24,25-epoxycycloartan-3-one (78) (3f3,24R)-24,25-epoxycycioartan- 3-01 (79) (3f3,24R)-24,25-epoxycycioartan-3-01 acetate (80) (23R)-3-oxolanosta-8,24-dien-23-ol (81) compound 82 fusidic acid (83) zeorin (84) 7f3-acetyl-22-hydroxyhopane (85) 7f3,22-dihydroxyhopane (86) oleanolic acid (87) erythodiol (88) 3-epioleanolic acid (89) oleanonic acid (90) lupeol (91) betulinic acid (92) betulin (93) epi-betulinic acid (94) lupeol acetate (95) lupenone (96) 3-hydroxynorlupen-2-one (97) 3-acetoxynorlupen-2-one (98) ursolic acid (99) uvaol (100) pomolic acid (101) pomolic acid acetate (102) tormentic acid (103) 2-epi-tormentic acid (104) euscaphic acid (105) niga-ichigoside Fl aglycone (106) Phytol, derivatives, and analogs (E)-phytol (107) (E)-phytyl acetate (108) (E)-phytol methyl ether (109) phytyl amine (110)
Source synthetic analog Montonoa grandiflora synthetic analog Euthamio leptocephela Magnolia acuminota Sigma-Aldrich Co.
MIC (,ug/ml)' >128 16 16 16 32 8
Tetradenio riporio juniperus excelsa Salvia multicau/is Salvia multicaulis Salvia multicaulis Salvia multicaulis Salvia multicaulis Salvia multicaulis Salvia multicaulis Azorella madreporica juniperus excelsa
25-100d 15.0b 6.0b 0.46' 5.6' 1.2' 2.0' 1.2' 0.89' 7.3' 20 14.4b
Ajuga remota synthetic analog Sigma-Aldrich Co. Melia volkensii Melia volkensii Melia volkensii Borrichia frutescens Borrichia frutescens Borrichia frutescens Borrichia frutescens synthetic analog Sigma-Aldrich Co. Sarmiento scandens Sarmienta scandens Sarmienta scandens Baceharis patagonica Baeeharis patagonica junel/ia tridens junel/ia tridens Chuquiraga ulicina Monttea aphylla Chuquiraga ulicina Chuquiraga ulieina Chuquiraga ulieina Chuquiraga ulicina Aspidosperma quebraeho-blanca Acaena pinnatifida Acaena pinnatifida Acaena pinnatifida Acaena pinnatifida Acaena pinnatifida Acaena pinnatifida
1 8 >128 16 4 16 8 8 >128 64-128 >128 4 8' >128' >128' 64' 64' 16 16 64' 32' 32' 64' >128' >128' >128' >128' 32' 32' 64' 32' 32' >128' 128' >128'
Lucas volkensii
synthetic analog
synthetic analog
synthetic analog
Planta Med 67 (2001) 687 Reference (31) (45) (26) (Robbs, S.L., 1997 unpublished) (26) (27) (33) (34) (34) (32) (32) (32) (32) (32) (32) (32) (35) (34) (36) (36) (36) (37) (37) (37) (25) (25) (25) (25) (25) (25), (46) (38) (38) (38) (38) (38) (39) (39) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (38) (27) (27) (26) (26)
688 Planta Med 67 (2001)
Table 1 cant.
Compound Class Name phytyl amine (110) phytyl diisapropylamine (111) (Z)-phytal (112) (3R,S.7R, 11 R)-phytanal (113) (E)-phytol epaxidation epimers (114) (3R.S,7R.ll R)-phytanic acid (115) 2-phytylphenal (116) phytantrial (117) 2-hexadecanol (118)
Source synthetic analog synthetic analog Sigma-Aldrich Co. synthetic analog synthetic analog synthetic analog synthetic analog Sigma-Aldrich Co. Sigma-Aldrich Co.
MIC tug/mil' 32 64 2 2 8 >128 32 16 8
" Biological activity determined using BAGEC radiorespirometric bioassay except where noted othen','ise. b Biological activity determined using disc-diffusion method. e Biological activity determined using broth microdilution method. d Biological activity determined by conventional proportion method. Clinical isolates of fd. tuberculosis were used in the bioassay. " Concentration reported in ,u1\t
Charles L. Cantrell et al. Reference (26) (26) (27) (27) (27) (27) (26) (26) (26)
Monoterpenes Due to the small number of monoterpenes with reported biological data and their structural similarity to phytol and its analogs, a more detailed structure-activity analysis will be discussed together with the results on phytol. It should be briefly pointed out, however, that among the open-chain monoterpenes, citronellol (1), nerol (2), and geraniol (3) gave MICs of 64,128 and 64,Ug!ml, respectively (Table 1) (26), (27). z- Compounds 2 and 3 represent respective and E-isomers, and 1 being saturated at C-2; this suggests that the presence of a C-2 double bond has only minor influences upon the activity.
~ "'" '3C/
~ OH
~ I"", OI""H , 3
as well as the sesquiterpenes obtained by reductive opening of the lactone ring (19, 20, and 21) (29) showed no activity against M. tuberculosis at concentrations below 128,Ug!ml, suggesting that the presence of the exocyclic o:-methylene-ylactone moiety is essential. but not sufficient, for activity.
~~ ~~~ 01 11--( r,,~ 1--( y~~
911flH. i3-dihydro
~9 ~l!t1I 'IQti=
12 R = H
13R=OH 14 R = OH; 11!lH. 13-dih,'dro
0 15 R = H 1GR=OH 17 R:::: OH: 11nH, 13-dihydro
Sesquiterpenes Among the over fifty sesquiterpenes, mainly sesquiterpene lactones of the germacranolide, guaianolide, and eudesmanolide type, tests resulted in MICs ranging from 2,Ug!ml to >128 ,Ug!ml. A series of o:-methylene~rlactonecontaining germacranolides showed MICs at or below 64,Ug!mL (28), The relatively lipophilic costunolide (4) gave an MIC of 32,ug!ml while its 4-epoxide derivative, parthenolide (6), showed an increased activity against M. tuberculosis (MIC 16,ug!ml). The 1(10)-epoxycostunolide (8) was less active than 4 and 6 with an MIC of 64,ug!ml, suggesting that the position of the epoxide within the medium ring has a distinct influence on the activity. With the exception of the non-active diepoxide 1,10-epoxyparthenolide (10), all the sesquiterpene lactones in this series with an o:-methylene-rlactone moiety (4, 6, 8, 13, 16, 12 and 15) have activities with MICs at 64,Ug!ml or below, In contrast, their 11I3H,13-dihydro derivatives (5, 7, 9,14, and 17)
21 Parthenolide, with an MIC of 16,Ug!ml, was the most active germacranolide tested and exhibited a higher MIC than costunolide (MIC = 32,Ug!ml). Moreover, 1(1O)-epoxycostunolide (8) and the diepoxide 10, although both with an o:-methylenerlactone moiety and an epoxide at C-1(1O), neither compound showed activity comparable with parthenolide (6). It has therefore been suggested that the higher antimycobacterial activity of parthenoIide (6) may be due to the presence of two sites of alkylation, one site being the o:-methylene-rlac-
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Planta Med 67 (2001) 689
tone moiety and the other site at C-lO, the electron deficient center being generated by a transannular cyclization with the C-1(10) double bond being the donor and the C-5 epoxide the nucleophilic receptor (28).
35. Also, tenulin (38) differs from 42 in its absence of an amethylene-y-lactone moiety. Consequently, its MIC of 128,ug/ ml further supports the necessity of this lactone group for significant activity.
A series of guaianolides screened against M. tuberculosis demonstrated MlCs ranging from 2 to >128 ,ug/ml, with the lipophilic dehydrocostuslactone (22) (MIC of 2,ug/ml) being the most active (30). Semi-synthetic mono- and diepoxide derivatives of 22 (25 -30), as well as its hydroxy analogs showed very distinct activity trends. Monoepoxides 25, 26, and 27 were more active than the more polar diepoxides 28, 29, and 30, which in turn were more active than the more polar hydroxy analogs 23 and 24. It has been suggested that the low MIC of 22 may be due not only to its exocyclic methylene lactone moiety but also to its lipophilic nature (30). This was supported by the decreased activity from 22 to the more polar monoepoxides via the diepoxides to the most polar hydroxy analogs 23 and 24. Micheliolide (31) (MIC 50llg/mL) holds a unique position, possibly due to the ability to undergo an intramolecular substitution involving the 1(10)-double bond as a nucleophilic donor and the C-4 hydroxy as a leaving group, thus allowing a double alkylation as in parthenolide (6).
The pseudoguaianolides damsin (33), parthenin (37), and aromaticin (42) showed antimycobacterial activity with MICs of 32,64, and 16Ilg/ml, respectively. This suggests that two alkylating sites within the molecule such as 42, increase activity. Again, the more polar C-8 hydroxy (34) and acetoxy (35) analogs of 33 gave MICs of 128 Ilg/ml, that is, much lower activity than 33, which may be due to the more polar nature of 34 and
rn R, .. nrr o<~ ~
22 RoR.=H 0
OH: = 23 R
R1 = H
24 R=H:R 1 =OH
25 0
26 R=P';;P~XY 27 R =a-=::poxy
",~ ""~
Bioassay-directed investigations of Inula helenium and Rudbeclda subtomentosa resulted in the isolation of eudesmanolides, with MICs ranging from 8 to >128 Ilg/ml. Moderate activities were observed for alantolactone (43), isoalantolactone (44), 11 aH,13-dihydroisoalantolactone (48), alloalantolactone (50), and 3-oxoalloalantolactone (51) (31). Compounds 43, 44, and 50 all gave MICs of 32 ,ug/ml, while 48 and 51 were not active (MICs >128 and 128Ilg/ml, respectively). Stereoselective epoxidation of 43 gave the eudesmanolide 5a-epoxyalantolactone (53) with an MIC of 8llg/ml while oxidation of 43 with OS04 resulted in the inactive dihydroxy derivative 54. Analogs of 44 include the monoepoxide 52, the C-2 hydroxy derivative 47, the conjugated ketone 45, and alcohol 46 with MlCs of 32,64,16, and 32 Ilg/ml, respectively.
As seen above with the various structural types of sesquiterpene lactones, the a-methylene~y-Iactonemoiety appears to be an essential, but not sufficient, structural requirement for significant activity. The necessity of the presence of an amethylene-y-Iactone group is supported by the moderate to high activity of a-methylene~y-Iactone bearing sesquiterpenes, when compared with the inactive 11aH,13-dihydro derivatives with values of 128 Ilg/ml or higher. The presence of a second alkylating site, such as an a,j3-unsaturated carbonyl group and/or an epoxide function together with moderate to high lipophilicity, seems to enhance the in vitro antimycobac-
~~0'OF=RR' 2~0~'FO = ~ fty0'o F=
44 R, = H
45 R, = 0: R2 = H; 1, 2-d.b = 46 R, = fl-0H; R2 H: 1, 2-d.b = 47 R, = H; R2 a-OH
50 R = H
51 RoO
R 28 = R1 a-epoxy: 2 = ~-epoxy
29 R, = a-ep':;lxy: R2::: u-epoxy
30 R, = (I-eODxy: R:; = u-epoxy
?C~!'N'"= 34 R:::: OH 0 35 R:::: OA:. 38
55 564.5-epoxy
I '"
H,C, /OH
58 ~OH
690 Planta Med 67 (2001)
Charles L. Cantrell et al.
terial activity. For instance, encelin (45) is more active than 44 and 5a-epoxyalantolactone (53), with an MIC of 8,ugfml, is significantly more active than its precursor 43 with an MIC of 32,ugfml. The sesquiterpenes curcuphenol (57), nerolidol (58), and farnesol (59) gave MICs of 16,32, and 8,ugfml, respectively. Compounds 58 and 59 are constitutional isomers differing in the positioning of the allylic alcohol. Due to the structural similarity of 57, 58, and 59 with phytol and its analogs, a more detailed discussion of their biological activities will follow later. Diterpenes A limited number of diterpenes have been tested for antituberculosis activity and some have demonstrated remarlc=tfZ\
/ -:;.. H R,
60 R, ::: CH 20H: R2 ::: OH
61 R1 ::: COOH; R: = H
~ rYY 63 R=H '.,64 R=Mi;
65 R=H 66 R=Me
&~o I "'"
"'" ""'0- 0
67 R=H 68 R=A::
,I h
\ ,-~:::
~ ~
Triterpenes and Sterols Numerous triterpenoids and sterols have been tested with MICs ranging from 1 to >128 ,ugfml. Bioassay-guided investi- gations of active fractions from Ajuga remota led to the isola- tion of the most active triterpenoid, ergosterol-5,8-endoperoxide (72), with an MIC of 1 flgfml (36). Acetylation of 72 provided the acetate, 73, with an MIC of 8,ugfml. In contrast, the parent compound, ergosterol (74), gave an MIC of>128 ,ugfml. Compound 72 was prepared in a one step synthesis from commercially available ergosterol (74) (36) making it an ideal candidate for future structure-activity relationship studies. Within this set of ergosterol derivatives, it appears that the presence of a C-3-0H combined with the endoperoxide group is necessary for high anti mycobacterial activity. Bioactivity-guided investigations of Melia volkensii resulted in the isolation of 12j3-hydroxykulactone (75), 6j3-hydroxykulactone (76), and kulonate (77). Compounds 75 and 77 both had MICs of 16,ugfml while compound 76 was more active with an MlC of 4,ugfml (37). From this set of limited data, it appears that a hydroxy group at C-6 gives rise to higher activity than a C-12 hydroxy group. Based on the same activity of 75 and 77, there appears to be little or no effect on biological activity upon methanolysis of the lactone moiety.
R 72 R::: OH 73 R 0,:".: 75 R,::: OH 76 R1 :::: H
,I I RI ~ HO 74
','~ OH oeH , i f , I I
78 R::: 0
79 R '"
80 R:::
In another bioassay-guided investigation, Borrichia frutescens afforded a number of antimycobacterial cycloartanes (25). The most active of these compounds were (24R)-24.25-epoxycycloartan-3-one (78) and (3j3,24R)-24,25-epoxycycloartan-3-
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Planta Med 67 (2001) 691
01 (79) with MICs of 8 pgjml, whereas the acetate 80 was inactive (MIC >128 ,ugjml). Unsuccessful attempts to oxidize 79 to 78 resulted in the synthesis of the inactive degradation product 82 (MIC >128 ,ugjml), and the isolate 81 gave an MIC of 64 -128 pgjml. Correlations of structural features and the MICs of these five triterpenes suggest that the presence of the C-3 keto andjor {:l-hydroxy group, the cyclopropane ring and the epoxide moieties, as in 78 and 79, seem to playa major role in the in vitro antituberculosis activity. Both, the cyclopropane and epoxide functions are absent in 81, resulting in the loss of activity. Also, the loss of activity by acetylation of the C-3 hydroxy group (MIC >128 ,ugjmL) strongly suggests that either a free hydroxy or a keto group at C-3 is required for significant activity. Several pentacyclic triterpenoids have been isolated with activities ranging from 8 to >128 ,uM. The most active of these compounds was zeorin (84), isolated by bioassay-guided fractionation of Sarmienta scandens and shown to have an MIC of 8 pgjml (38). 7{3,22-Dihydroxyhopane (86) contains a {3-hydroxy group at C-7 rather than an a-hydroxy at C-6 in 84, resulting in a loss of activity (MIC >128 ,uM). In addition, the C-7 acetoxy derivative of 86, 7{3-acetyl-22-hydroxyhopane (85), was inactive (38). 3-Epioleanolic acid (89) and oleanonic acid (90), which differ in the functional group present at C-3, were both isolated from]unellia tridens and shown to have MICs of 16pgjml (39). Compound 89 contains an a.-hydroxy group at C-3 while 90 bears a ketone. Oleanolic acid 87 differs from 89 by the presence of a C-3 {3 rather than an a-hydroxy group, resulting in a less active derivative with an MIC of 64 ,uM. The dihydroxy analog of 87, erythrodiol (88), gave a similar MIC value of 64 pgjml. Similarly, ursolic acid (99) and betulinic acid (92) gave MIC values of 32 ,uM, identical to those of uvaol (100) and betulin (93) (38). In contrast to the results obtained when comparing the MlCs of compounds 87 and 89, betulinic acid (92) with its {3-hydroxy group at C-3 is more active than its C-3 epimer, epi-betulinic acid (94) (MIC 64 pM). A!so, re-
84 R1 ::: OH: R;;:::: H 85 R j :::: H: Rz ::: OAs 86 R, = H: R, = OH
87 R1 = ~-oH: Rz = eOCH = 88 R, = Il-OH, R, GH,OH 89 R1 ::: u-OH: R2 = eOOH = 90 R1 = 0: Rz eOCH
placement of the 3-hydroxy group of lupeol (91) with either an acetate or a ketone leads to complete loss of activity in lupeol acetate (95) and lupenone (96). A similar result was observed for the C-3 acetoxy derivative 80 when compared to 79. However, the 3-oxo derivative 78 gave the same MIC as 79 which is in contrast to the correlations mentioned above. Further complicating the situation was the observation that pomolic acid 3-acetate (102) was more active than pomolic acid (101) itself. Comparison of ursolic acid (99) with pomolic acid (101), tormentic acid (103), 2-epi-tormentic acid (104), euscaphic acid (l05) and the aglycone of niga-ichigoside (106) showed that additional hydroxy groups in rings A and E can, in some cases, reduce activity (38). These conflicting results observed among the various groups of triterpenoids makes it difficult to predict structural requirements for antimycobacterial activity. Phytol, Derivatives and Analogs This group of open chain diterpenes covers phytol, its derivatives and structural analogs. Also included are the previously mentioned linear monoterpenes 1, 2, and 3 and diterpenes 57, 58, and 59. Within this set of structural analogs, all but two compounds showed MICs at or below 64pgjml. One of the most active compounds in this series, (E)-phytol (107) (MIC 2 pgjml) was isolated from Lucas volkensii, using a bioassayguided fractionation (27). In addition, the analogs (Z)-phytol (112) and (3R,S,7R,11R)-phytanol (113) demonstrated MICs of 2 pgjmi, suggesting that the 2,3-double bond may not be essential for bioactivity. However, (E)-phytyl acetate (108) and (E)-phytol methyl ether (109) showed MICs of 16,ugjml implying that a free hydroxy group, as present in 107, is required for significant activity. Due to their structural and biosynthetic similarities to (E)-phytol, geraniol (3) and farnesol (59) were also tested against M. tuberculosis. The monoterpene alcohol geraniol had an MIC of 64,ugjml while the more lipophilic farnesol with a 15-carbon chain gave a significant increase in activity with an MIC of 8 pgjml. Citronellal (1) and nerol (2) were tested due to their structural similarity to geraniol and gave MICs of 64 and 128pgjml, respectively. The identical MlCs of 64,Ugjml between geraniol and citronellol further support the observation above for phytol and its reduced isomer (3R,S,7R,11R)-phytanol that the 2-double bond is not essential for biological activity. Additional derivatives and analogs tested include curcuphenol (57), nerolidol (58), phytylamine (110), phytyldiisopropylamine (111), (E)-phytol epoxidation epimers (114), (3R,S,7R,11R)-phytanic acid (115), 2-phytylphenol (116), phytantriol (117), and 2-hexadecanol (118) giving MICs of 16,32, 32, 64, 8, >128, 32, 16, and 8 pgj mi, respectively (27), (26). It is significant to note that a complete loss of activity is found when the C-1 hydroxy of (3R,S,7R,11R)-phytanol (113) is oxidized to the carboxylic acid, (3R,S,7R,11R)-phytanic acid (115), again suggesting that the free hydroxy is essential for high activity.
91 R1 = j3·0H; R;;: ::: M&: RJ ::: CH2 92 R1 ::: j3-0H: R2 = eOCH: RJ ::: CHz 93 R, = poOH: R, = GH,OH: R, = GH, 94 R1 ::: c.-OH: R2 = eaOH: RJ ::: CH2 95 Rj = 0-0Ac: R2 ::::: r\iie: R3 ::: CH2 96 R, = 0 R, = Me: R, = GH, 97 R1 = p·OH: R2 ::: Me: R3 = 0 98 R, = :',-OAe: R, = Me R, = 0
99 R1 ::: 13·0H; R2 ::: COOH; R3 = H; R.;::: Me; Rs = H 100 R, ::: 0·0H; R2 = CHzOH: RJ = H: R.;::: Me; Rs ::: H 101 R, = poOH: R, = GOOH: R, = OH: R, = Me: R, = H = = 102 R1 = (}..QAc; R2 ::: eOOH: R3 OH; R., Me: Rs = H 103 R1 = p-DH: R2::: eOCH; R:l = OH: R.;::: f-,c1e; Rs ::: c.:·OH 104 R t = P-OH; R2 = eOCH; R3 = OH; R.; = r.1e: Rs ::: 0-0H = = = = 105 R 1 ::: u-OH: R2 ::: eOCH; R:; = OH; R,;::: t.1e: Rs = a-0H 106 R1 ::: j3·0H; R2 eOGH: R3 GH: R.: CH20H: Rs c:-OH
The most interesting observation among the above group of compounds is the relationship between the calculated log P values and the MIC. Higher lipophilicity generally results in higher antimycobacterial activity, within a series of structurally similar compounds. For example, the more lipophilic 20carbon phytol (log P = 8.66) is more active than the 15-carbon farnesol (log P = 5.31) which is more active than the 10carbon geraniol (3) (log P = 3.28). However, there appears to
692 Planta Med 67 (2001)
Charles L. Cantrell et al.
107 R=OH 108 R=OAc 109 R=OCH, 110 R=NH, 111 R=N(isopropy1h
113 114 115 116 117
70 , - - - - - - - - - - - - - - - , 60 , - - \ - - - - .. 50
20 } - - - - - - - \ - - - - · - - - - I - - - - } 10 +----_. --
Log p.
Fig.l The relationship between calculated Log P values and antimycobacterial activity of phytol, its derivatives, and structural analogs. Plotted compounds include: 1, 3, 57. 58, 59, 107, 108, 109. 111, 112, 113, 114, 116, 117, 118. Curve fitting was performed using a polynomial fourth order equation.· Log P values were estimated using ACD Labs Log P module (47).
ity was found in the (E)-phytol series in which the highest activity was observed for the least polar 20-carbon (E)-phytol (107) followed by the lS-carbon analog faroesol (59), which was more active than the more polar lO-carbon geraniol (3).
OH 118 be a point at which further increasing the lipophilicity results in lower antimycobacterial activity (Fig.l). Replacing the hydroxy of phytol with nitrogen containing functional groups strongly reduces activity in spite of an increase in lipophilicity. For example, replacing the hydroxy of phytol with a diisopropylamine as in 111 (log P = 11.14) results in a more lipophylic molecule that is less active. Figure 1 is a plot of calculated log P values versus their respective MlC's, for a selected group of phytol derivatives and analogs. Conclusions A broad range of plant terpenoids from various classes have been evaluated for their in vitro antimycobacterial activity. The most active terpenoid presented in this review is the oorditerpenoid 12-demethylmulticauline (63), isolated from the roots of Salvia multicaulis, with an MlC of 0.46,ug/ml. It is more active than the first line tuberculosis drug ethambutol and nearly as active as rifampin. Further terpenoids with high activities are the sesquiterpene dehydrocostuslactone (22) with an MIC of 2,ug/ml, the sterol ergosterol-S,8-endoperoxide (72) (MIC 1,ug/ml), and (E)-phytol (107) with an MIC of 2,ug/ml. Distinct similarities between different classes of terpenes and the structural features important for high activity within each class have been observed. In each series it was shown that more lipophilic compounds are significantly more active than their more polar analogs. This was observed for the sesquiterpene lactone dehydrocostuslactone (22) which gave an MIC of 2,ug/ml, when compared to its C-3 and C-7 hydroxy analogs, both with MICs of>128 ,ug/ml. Furthermore, higher MICs were found for the monoepoxide derivatives of 22, followed by even higher MICs of the diepoxides. Additional support for a correlation between lipophilicity and antimycobacterial activ-
Among phytol (107), its acetate (108), and methyl ether analog 109, both derivatives gave distinct decreases in activity. Also, a decrease of activity was found for the more lipophilic nitrogen containing phytol analogs 110 and 111. Similar relationships were observed among the triterpenes and sterols where a free hydroxy or carbonyl at C-3 appears to be essential for high activity. For instance, compounds 72 and 79 were both more active than their corresponding C-3 acetoxy derivatives, 73 and 80, respectively. The resurgence of infectious diseases such as drug-resistant tuberculosis necessitates more intense future efforts in the discovery of new specific drugs from natural and synthetic sources. The recent development of efficient and reproducible bioassays plays a significant role in the present and future discovery and development of new anti-TB leads. A number of compounds covered in this review possess in vitro antimycobacterial activities comparable to standard anti-TB drugs. They certainly warrant further investigations on the long path from the initial activity findings to the development of new antituberculosis drugs. Acknowledgements The authors are grateful to Dr. Mohamed S. Rajab from the Department of Chemistry, Moi University, Eldoret, Kenya for helpful discussions. References 1 Dye C, Scheele S, Dolin P, Pathania V, Raviglione Me. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. journal of the American Medical Association 1999; 282: 677-86 2 Raviglione MC, Snider DE, jr., Kochi A. Global epidemiology of tuberculosis. Morbidity and mortality of a worldwide epidemic. journal of the American Medical Association 1995; 273: 220-6
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20 Ji B, Lounis N, Maslo C, Truffot-Pernot C, Bonnafous P, Grosset]. In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 1998; 42: 2066-9 21 Miyazaki E, Miyazaki M, Chen JM, Chaisson RE, Bishai WR. Moxifloxacin (BAY12 - 8039), a new 8-methoxyquinolone, is active in a mouse model of tuberculosis. Antimicrobial Agents and Chemotherapy 1999; 43: 85-9 22 Cynamon MH, Klemens SP, Sharpe CA, Chase S. Activities of several novel oxazolidinones against Mycobacterium tuberculosis in a murine model. Antimicrobial Agents and Chemotherapy 1999; 43: 1189-91 23 Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, Anderson SW, Towell JA, Yuan Y, McMurray DN, Kreiswirth BN, Barry CE, Baker WR. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000; 405: 962-6 24 Collins L. Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 1997; 41: 1004-9 25 Cantrell CL, Lu T, Fronczek FR, Fischer NH, Adams LB, Franzblau SG. Antimycobacterial cycloartanes from Borrichia frutescens. Journal of natural products 1996; 59: 1131- 6 26 Cantrell CL. Antimycobacterial natural products from higher plants. Department of Chemistry, Baton Rouge, LA: Louisiana State University 1998: 173 pp 27 Rajab MS, Cantrell CL, Franzblau SG, Fischer NH. Antimycobacterial activity of (E)-phytol and derivatives. A preliminary structure-activity study. Planta Medica 1998; 64: 2-4 28 Fischer NH, Lu T, Cantrell CL, Castaneda-Acosta J, Quijano L, Franzblau SG. Antimycobacterial evaluation of germacranolides. Phytochemistry 1998; 49: 559-64 29 Lu T, Fischer NH. Spectral data of chemical modification products of costunolide. Spectroscopy Letters 1996; 29: 437 -48 30 Cantrell CL, Nunez IS, Castaneda-Acosta J, Foroozesh M, Fronczek FR, Fischer NH, Franzblau SG. Antimycobacterial activities of deliydrOcostus lactone and its oxidation products. Journal of Natural Products 1998; 61: 1181 - 6 31 Cantrell CL, Abate L, Fronczek FR, Franzblau SG, Quijano L, Fischer NH. Antimycobacterial eudesmanolides from Inula helenium and Rudbeckia subtomentosa. Planta Medica 1999; 65: 351- 5 32 Ulubelen A, Topcu G, Johansson CB. Norditerpenoids and diterpenoids from Salvia multicaulis with antituberculous activity. Journal of Natural Products 1997; 60: 1275 - 80 33 Puyvelde LV, Ntawukiliyayo JD, Portaels F. In vitro inhibition of mycobacteria by Rwandese medicinal plants. Phytotherapy Research 1994; 8: 65-9 34 Topcu G, Erenler R, Cakmak 0, Johansson CB, Celik C, Chai H-B, Pezzuto JM. Diterpenes from the berries of Juniperus excelsa. Phytochemistry 1999; 50: 1195-9 35 Wachter GA, Franzblau SG, Montenegro G, Suarez E, Fortunato RH, Saavedra E, Timmermann BN. A new antitubercular mulinane diterpenoid from Azorella madreporica Clos. Journal of Natural Products 1998; 61: 965 - 8 36 Cantrell CL, Rajab MS, Franzblau SG, Fronczek FR, Fischer NH. Antimycobacterial ergosterol-5,8-endoperoxide from Ajuga remota. Planta Medica 1999; 65: 732-4 37 Cantrell CL, Rajab MS, Franzblau SG, Fischer NH. Antimycobacterial triterpenes from Melia volkensii. Journal of Natural Products 1999; 62: 546-8 38 Wachter GA, Valcic 5, Flagg ML, Franzblau SG, Montenegro G, Suarez E, Timmermann BN. Antitubercular activity of pentacyclic triterpenoids from plants of Argentina and Chile. Phytomedicine 1999; 6: 341 - 5
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Dr. Charles L. Cantrell USDA, ARS National Center for Agricultural Utilization Research 1815 North University Street Peoria Illinois 61604 U.S.A. E-mail: [email protected] Fax: +1-309-681-6686 Tel.: +1-309-681-6349
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