Accepted Article View metadata,citationandsimilarpapersatcore.ac.uk Moreover, the This by is protectedarticle reserved. Allrights copyright. doi: 10.1002/cbdv.201700118 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted for publication andundergone peerfull but review not has , reaching as far as Venezuela, while , Bolivia and northern . One single ( The Introduction Full Paper Article Type: Keywords Miers, based mostly on morphological characters. Recent molecular phylogenies in proved that showed moderate activity (IC carcinoma cell lines (PC3 and LNCaP) and two human breast cancer cell lines (MCF-7 and T47D). Several compounds morphologically close to ramosissima salpichrolides A, C, I, S, and anunreportedwithanolide named salpichrolide V ( glandulosa take the forms of aof thetake scandent forms shrub ( c Departamento deQuímica Orgánica, Universidad de Buenos Aires, Unidad de Microanálisis y Métodos Físicos Aplicados ala Chemotaxonomic Considerations and Biological Evaluation in Córdoba, CC 495, 5000 Córdoba, Argentina, [email protected] (V. Nicotra), [email protected] (G. Barboza) a Química Orgánica (UMYMFOR-CONICET), Facultad de Ciencias, Exactas y Naturales, Ciudad Autónoma de Buenos Aires, Facultad de Ciencias Químicas, Instituto Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Universidad Nacional de Phytochemical Study of theGenus Ana ValentinaBasso, Twelve Twelve D’Arcy Careaga, taxa is in agreementwith molecular studies, which suggest that : Salpichroa subsp. Salpichroa , [ 3 S Salpichroa ] and Hunziker in vitro . tristis d glandulosa Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina b Museo de Historia Natural, Universidad Privada Antenor Orrego, CC 1075, Trujillo, Perú var. var. (Solanaceae)Miers comprises around 17 species, restricted mainly to the highland Puna and Prepuna of cytotoxic activitycytotoxic natural of a set of salpichrolides andderivatives wasexamined twoagainst prostate c,d • Withanolides • Structure elucidation • Chemotaxonomic markers •Cytotoxicity taxa have been phytochemically analyzed. From aerialthe parts of Salpichroa Juan Carlos Calvo, Juan tristis [ 1 50 ] Prostate andBreast Cancer Cells. , placed Salpichroa together with = 64.91 = S. glandulosa , and a Segundo LeivaGonzález, , are, distant in the systematic of the Solanoideae subfamily. Figure 1A, D S. weberbauerii S. − 29.97 S. origanifoliaS. subps. μ M). ), ora hanging shrub ( weddellii d PaulaA. Sacca, , nowithanolides were found. The chemical content of 85 ca. % of the (Lam.) Baillon has aworldwide distribution. S. tristis Argentina , S. leucantha S. Nectouxia

Version of Record. Please Version as this cite ofRecord. article Miers) occurs Miers) throughouthighland thegrasslands the in b Gloria Estela Barboza,*Estela Gloria Figure E 1C, Kunth and Kunth Salpichroa Salpichroa , S. micranthaS. d and Viviana EstelaNicotra* 1 ), were isolated. In Jaborosa ), or a), or procumbent( shrub and , S. microloba Jaborosa S. scandensS. Juss. in the tribe Jaboroseae (Solanaceae). (Solanaceae).

S. dependens , a, genus considered [ 1,2 , ] These species can S. proboscidea , the four known ,a Valeria Pilar Valeria Salpichroa Figure 1B, F , S. gayi S. ,a

belongs belongs , S. , S. ). ). brought toyouby provided byCONICETDigital CORE Accepted Article micrantha (with two subspecies: taxa is herein reported. The species studiedwere: reviews. and commonly known aswithanolide the skeleton, with their chemistry and occurrence having been the subject of several This by is protectedarticle reserved. Allrights copyright. to adifferent and distant clade from arrangements,which can be considered differentat hierarchical levels chemotaxonomic markers. Solanoideae subfamily. Some clades of the subfam. Solanoideae also contain withanolides with exclusive interesting structural agreement with the molecular proposal to place S compounds exhibit interesting biological activities assuch antifeedant, effects. membered salpichrolides. They have been isolated from . tristis Withanolides are steroidal lactones built on an ergostane skeleton of 28 carbons functionalized carbons at 1, 22, and26, As part of an ongoing program aimed at to clarify the systematic position of [ 19 Miers [ 6-8 ]

Benoist, ]

750Over withanolides have been described, principally, notbut exclusively, generafrom belonging to the aromatic ring-D and a family of these related ergostane derivatives constitute animportant group called

var. tristis tristis S. microlobaS.

subsp. (three samples),and glandulosa S. Keel, Jaborosa and subsp. S. proboscideaS. S. origanifoliaS. . S. weberbauerii S.

[ 4,5 Salpichroa ] Until now Until the phytochemical evidence was quite fragmentary, but in S. dependensS. weddellii Benoist, and and (Benoist) Keel), Dammer Jaborosa S. tristis (Hook.) Miers, S. ramosissima [ 15-17 . var. inindependent clades. ] antiproliferative, lehmannii S. leucantha S. gayi Salpichroa Miers, (Dammer) Keel. Benoist, S. scandens [ Pereyra, Quip.Pereyra,Leiva, &S. 18 , aphytochemical study of twelve ] and cancer chemopreventive S. glandulosa S. [ 6 [ 6 ]

] Withanolides with asix- Dammer (two samples), samples), (two Dammer [ 9-14 ] Many of these (Hook.) Miers S.

Accepted Article This by is protectedarticle reserved. Allrights copyright. 1. Figure ramosissima Salpichroa . E. . E. S . didierana speciesnatural intheir habitat. A. . F. S . microloba . S . glandulosa subsp. subsp. glandulosa . B. . B. S . gayi . C. S . weberbaverii . D. . D. S .

Accepted Article generawithin subfam. Solanoideae and in most species of some of these genera (eg. This by is protectedarticle reserved. Allrights copyright. purification yieldto newthe compound of parts aerial the of extract dichloromethane The Phytochemical study. andResults Discussion and Figure 2. the signal of H -C (6) with the signals of C (5), C (10) ( C and shown yield to the salpichrolides A, C, S, and V ( cross-correlation peak observed between H -C (6) and H -C (9) ( the chlorine group was established through the multiplicity and coupling constant value of H -C (6) ( chain epoxy lactolarrangement. Regarding the A and B rings, the corresponding to a salpichrolide-typewithanolide characterized by the aromatic ring-D and a 77.5 and 64.0 in the presence of a chlorine substituent at C (6) position. The substitution pattern inring B was further confirmed by the signals a signal at δ

genera. have been reported from the withanolides found in other species of Solanaceae. To date, at least thirty-five withanolideswith chlorine substituent on ring 5 Information). Thus, the structure of compound spectrum displayedspectrum the signals characteristic correspondingaromaticprotons to at salpichrolide V ( (25) methyl groups at C (24) and C (25) at the oxymethine proton at (15), H -C (16), and H -C (18), respectively; (ii) the presence of an epoxy was lactol evidenced by the signal corresponding to

α 28 5.91 ( ,26-dihydroxy-17(13 H 3 2 ] 1 , 35 OR H NMR but no withanolides were detected from any of these. Withanolides have been reported in the majority of the The dichloromethane and ethyl acetate extracts of the remainder eleven δ ClO 91.7 91.7 [ dd O O 1 6,8,20 4 Withanolides isolated from δ HO 5 , 5 4.07 ( 19 Na. FromNa. the following observations, the NMR spectroscopic data of compound J ] 10 Following asimilar procedure, the dichloromethane extract of =10.2 and 2.8 Hz) and 6.57 ( [ C (26) Cl 6 9 11 1 t 12 , ) showed a quasimolecular ion [M+Na] 7 J 8 apcrld S salpichrolide apcrld ( V salpichrolide 13 = 2.9 Hz), bearing in mind that the molecule has a chlorine atom by HRESITOFMS, suggested the ] 13 , 14 δ C NMR spectrum, assigned to C (5) and C (6), respectively, and by the cross-correlation peaks between 15 18 16.9 → 18) abeo-5 δ 17 4.92 ( 4.92 16 [ 21 Jaborosa C (27) 22 20 O Salpichroa 24 d 23 28 , O O J ] , = 9.9 Hz), assigned to the hydrogen H -C (26), and also by resonancesthe of the two 1 α 26 δ O δ OH ) -ergosta-2,13,15,17-tetraen-1-one. Compound , 1, and 19.1 1.31 (H 25 Nicandra and the known salpichrolides A, OH 27 species. species. ddd HO HO , 1 3 J was established as (20 R= -27) and 1.29 (H O [ Adans., = 10.2, 5.1, and 2.2 Hz), typical of a1-oxo-2-en system inring A, while the S. scandens C (28) OH O -glc 1 δ ) by comparisonwith published spectroscopic and physical data. OH O 52.9), and (8)( C + ] at at , observed , inthe apcrld A salpichrolide m/z fromwas(sample Argentinasubjected 1) chromatographic to 509.2066, corresponding to an elemental offormula δ 3 -28), respectively;(iii) the signalsat 2.16) inthe NOESY experiment (see Supporting 1 L., L., H NMR spectrum of of Hspectrum NMR Tubocapsicum S ,22 δ [ 13 32.9) in the HMBC spectrum. The 13 S. scandens ] O C, C NMR spectrum, were agreementin with a side R ,24S,25 O [ Salpichroa 14 ] I, OH [ δ 11 7.14, ] (Wettst.) Makino, and 1 and S S Datura Datura is one of the examples of chlorinated ,26 O from Bolivia (sample 2) was analyzed 1 O exhibited olefinic protontwo at signals taxa studied were taxastudied analyzedby TLC HO R O δ 1 )-6 6.94, and [ revealed the typical signals 9 δ ] L., L., ( -lactol side chain: (i) the OH β Figure 2 Figure -chloro-22,26:24,25-diepoxy- Jaborosa apcrld C salpichrolide t , apcrld I salpichrolide J δ = 2.9 Hz), and by the δ 65.2 65.2 ). 5.91, assigned to H -C

HRESITOFMS of , Physalis, Withania [ C (24) C β orientation of O ] O Pauquy ,

δ O 1 64.0 H NMR H NMR OH OH OH B t δ [

C

Accepted Article miehei derivatives tested ( dependent), and on two human breast cancer cells that possessing the estrogen receptor, MCF-7 and T47D. The salpichrolide derivatives was examined on twohuman prostate carcinoma cell lines, PC-3 (androgen independent) and LNCaP (androgen negative cell lines shown by salpichrolide theA, Considering the selective antiproliferative activity toward hormone-dependent ER(+) breast cell lines when compared to ER(-) Citotoxic activity. clades. species to date.species to system (salpichrolide A and atropurpureum members of our working group conducted a phytochemical study in two This by is protectedarticle reserved. Allrights copyright. Withania features. 10-65 10-65 mentioned above in agreement with the molecular position of maintaining ring-D withanolides, with these latter

and salpichrolide T and compound exclusive rearrangements (trechonolides, sativolides, spiranoid- Salpichroa expansion. far from from far var. the unique species of cytotoxic activity, in agreementwith results previously reported in the literature. salpichrolide A and salpichrolide C showed the that cleavage of 5,6-epoxy-group the give to 5 the Solanaceae genera but only a few species of position ( Salpichroa The suprageneric position of Chemotaxonomical considerations. Salpichroa inhibitory concentration(IC withanolides, and 15,21-cyclowithanolides). well supported small clade, while reconstruction for 4 sensitive to the compounds tested. The compounds salpichrolides A, G, D, V,M, 2,3-dihydrosalpichrolide B, and the derivatives was used as reference anticancer drug. Overall, datathe obtained showed that the LNCaP and T47D cell lines were more and lehmannii) Previous phytochemical studies involving thirteen 11 μ (CECT 2749) and M. MTS one MTS M. step colorimetric assay was done to test the cytotoxic potential and to determine IC Jaborosa ). [ showed cytotoxic activity against at oneleast of the assayed cell lines (IC α 31 [ or 6 species, more accurateconclusions can bemade. In along with along species would be not so strange. ] [ Finally, no of loss activity was observed when the 5 28 ] However, in other genera such as ] Based on these different chemical contents found for each genus, we reaffirm phytochemicalthe support β Schrank), but Schrank), nowithanolides were found in any of them. ) led ato loss of activity (LNCaP and T47D), this is observed when comparing the compounds salpichrolide A have supported molecularthe proposal. [ 21-24 in the andsmall informal “Salpichroina clade” Jaborosa 2 ]

In addition In to consulting the published papers related to phytochemical ofstudies the − Nectouxia

Jaborosa 11 ) were obtained by biotransformation of salpichrolides A, C, and G with filamentous fungi Cunninghamella elegans 50 ; in; their study eighteen 1 ), are), summarized inTable Cell lines1. were exposure 48 for hours with the compounds at a range Salpichroa ). On the contrary, this structural difference enhanced cytotoxic activity against PC-3 cell line. and ( N. formosa Jaborosa 6, 6,

Nectouxia compounds possibly being involved in the biosynthetic pathway leading ring-D to in agreement with results previously reported for compounds with similar structural within subfam. Solanoideae is still unresolved. Although the traditional position groups was sister to the Eurasian genus [ 6 Knuth) were included. Once again, ] In contrast, only 17.5 % of the the of % 17.5 only contrast, In Jaborosa in the tribe Jaboroseae, Solanum in vitro (CECT 2113), Jaborosa and cytotoxic activity of a set of natural salpichrolides ( [ 6,9-14 Jaborosa L. (ca. 1500 species), withanolides have been reported only in two ] Salpichroa Now,with new knowledge concerning approximately 85 % of the species,three α γ [ ,6 4,5 Jaborosa -lactones, spiranoid withanolides at C-22, aromatic ring-A (Figure 4) speciestwo and α ] These molecular Thesestudies encompassed majorityof the the -epoxy functionality was replaced by 5 . Recently, Moré et al. [ 1,3,26 Solanum 25 Jaborosa , , all the analyzed species contained withanolideswith Therefore, the absence of withanolidos in some ] . molecular studies place Salpichroa [ Salpichroa 29 Atropa ] The expressed results, as half maximal [ 18,30 Salpichroa species ( species Salpichroa 50 ] and The presence of a hydroxyl group C-12at L. (tribe Hyoscyameae Endl.). = 64.91 = taxa (two species and one variety) and taxa contained aromatic or oxygenated Salpichroa S. comptumS. and [ 27 − taxa (S. 29.97 ] proposed aphylogenetic Nectouxia α ,6 distant in independent β Salpichroa μ -diol abolished the origanifolia C.V. and Morton M). ComparisonM). of 50 doses. Doxorubicin Figure 3 α Solanum were placed ina -chloro,6 and and ) and Rhizomucor species, species, β Nectouxia -hydroxy -hydroxy S. tristis S. S.

Accepted Article This by is protectedarticle reserved. Allrights copyright. > 65 44.06 ± 1.08 >65 >65 > 65 > 65 >65 >65 >65 >65 10.02 ± 0.01 > 65 6.11± 0.02 > 65 >65 >65 >65 64.63 ± 1.05 1.89 ± 0.01 >65 >65 1.63 ± 0.01 > 65 >65 >65 >65 >65 29.97 ± 1.02 >65 >65 a >65 >65 >65 doxorubicin > 65 > 65 >65 11 >65 >65 10 > 65 > 65 51.45 ± 1.06 9 > 65 >65 8 > 65 > 65 64.91 ± 1.03 7 > 65 6 > 65 45.25 ± 1.07 5 > 65 4 > 65 > 65 > 65 54.16 ± 1.03 41.79 ± 1.08 3 40.23 ± 58.68 ± 1.08 1.03 > 65 41.81 ± 1.08 2 > 65 > 65 > 65 >salpichrolide 65 V(1) 61.82 ± 1.04 > 65 55.69 ± 1.03 salpichrolide T 51.16 ± 1.05 44.61 ± 1.03 > 65 48.05 ± 1.08 salpichrolide S > 65 > 65 52.72 ± 1.04 > 65 salpichrolide M > 65 > 65 salpichrolide G 47.87 ± 1.03 salpichrolide D > 65 46.95 ± 1.06 salpichrolide C > 65 2,3-dihydrosalpicrolideB salpichrolide A breast and prostate tumor celllines. Table 1. Compound LNCaP LNCaP Compound PC-3 MCF-7 T47D IC 50 values given in

In vitro cytotoxic activity of salpichrolides A, C, D, M, G, S, T, and2,3-dihydrosalpichrolide Band derivatives μ M (mean ± S.D.)were obtained onthe basis of triplicate assay. a

1

−

11 againsthuman solid Accepted Article This by is protectedarticle reserved. Allrights copyright. Figure 3. HO HO O OR R= O O OH Salpichrolides assayed against human prostate and breast cancer celllines. O -glc O OH O apcrld A salpichrolide apcrld D salpichrolide apcrld S salpichrolide O O O O O O O OH OH OH OH O O O O O O ,-iyr apcrld B salpichrolide 2,3-dihydro O O OH apcrld G salpichrolide doxorubicin apcrld T salpichrolide OH OH OH O O OH OH O O O O O O O OH NH OH OH OH 2 O O O HO HO O Cl OH apcrld C salpichrolide apcrld M salpichrolide apcrld V salpichrolide O OH O O O O OH OH OH OH

Accepted Article This by is protectedarticle reserved. Allrights copyright. Figure 4. AcO AcO unnhmlaeeasRiouo miehei Rhizomucor elegans Cunninghamella HO HO Salpichrolide derivatives obtained by biotransformation HO O O OH OH O HO HO O O O OH OAc apcrld C salpichrolide 10 9 R=Ac R=H hzmcrmee hzmcrmiehei Rhizomucor miehei Rhizomucor Ac Ac 2 2 3 4 2 o/Py o/Py O O O O O OH O O O O O OH O OH OH OAc OR OAc 29 apcrld A salpichrolide assayedagainst human prostate andbreast O O O O O O O OH HO HO O O O OAc OH R apcrld G salpichrolide OH OAc OH

cancer cell lines. lines. cell cancer 6 5 11 7 Ac 8 OH O 2 o/Py O O O O O O O O O O OH OH OAc OH OH

Accepted Article been isolated from the generic level for two reasons: the aromatic ring-D withanolides are not exclusive of the Solanoideae subfamily. Moreover, salpichrolides should be considered to berelative taxonomic markers for 13º12’41.6’’S, 71º38’02.7’’W, Cuzco, Perú, in January 2012 (voucher under GB3411); 11º35’13.4’’S, 76º37’31.5’’W, Lima, Perú, in January 2012 (voucher under GB3387); This by is protectedarticle reserved. Allrights copyright. 2078) and these two has species also been deposited at CORD, and was identified by G. Barboza (GB); tristis Quillacollo, 17º27’06.6’’S, 66º21’11.6’’W, Cochabamba, Bolivia, inFebruary 2012 (voucher under GB 3615); km 89/90, 26°42’12.9’’S, 65°47’58’’W, Tucumán, Argentina, in April 2011(voucher under GB 3042); 11º50’42.7’’S, 76º22’57.2’’W, Lima, Perú, in January 2012 (voucher under GB3370); in Pisaq, 13º25’11.2’’S, 71º50’58.6’’W, Cuzco, Perú, in January 2012 (voucher under GB 3404); samples werecollected: Universidad Nacional de Córdoba (Argentina), and was identified by G. Barboza (GB) and LeivaS. González. The following A vouchertheall specimen for Material on aBruker AVANCE II AV-400 NMR spectrometer operating at 400.13 MHz for spectrophotometer, and the IR wasspectrum produced using a Nicolet 5-SXC spectrophotometer. NMR spectra were recorded Optical rotation was measured on a JASCO P-1010 polarimeter. The UV spectrum was obtained using a Shimadzu-260 Procedures General Experimental Section Experimental Thethechemical 85%ca. content of of Conclusions Solanum comptum weberbauerii tristis tristis Rayampampa, 08°01’16.9’’S, 78°39’44’’W, La Libertad, Perú, in January 2013 under(voucher GB 3948); 66º29’41.6’’W, Oruro, Bolivia, in March 2012 (voucher under GB 3630); Perú, in January 2012 (voucher under GB3428); (voucher under 3431);GB 2012 (voucher under 3417);GB January 2013 (voucher under GB 3949); relationshipwithanolides. studiesforof type this bands were visualized using ultraviolet light; (iii) compounds were from eluted the silica using CH performed under the following conditions: (i) the amount of sample appliedwas approximately 15 mg for 20 plate; cm (ii) the

(COSY, HSQC, HMBC and NOESY) were obtained using standard software. Chemical areshifts given in ppm ( 20, and preparative TLCwas carried out using gelsilica 60 F chromatographic separations were performed by column chromatography onsilica gel 60(0.063-0.200 andmm) Sephadex LH- TMSfrominternal standard. the HRESIQTOFMS TOFwas a Micro determined Daltonics II Bruker on spectrometer. The compounds in presentthe workwas moderate (IC because these metabolites are only present in 17.5% of the

Regarding the bioactivity of the salpichrolides and derivatives, although the cytotoxic activity observed for testedthe (sample 1) in Tafi, 75/76,km Tucumán, Argentina, in December 1995 (voucher under A. Hunziker T. 25544); (sample 3) in Carrasco, 17º28’30.4’’S, 65º38’14.9’’W, Cochabamba, Bolivia, in March 2012 (voucher under GB 3621); (sample 2) in Tafí, Mirador del Infiernillo, Tucumán, Argentina, in March 2006 (voucher under GB 1711); S. atropurpureumS. Dammer inQuello-Quello, 13º23’30’’S, 71º49’01’’W, Cuzco, Perú, in January 2012 (voucher under GB 3415). and Nicandra physalodes S. atropurpureum S. Salpichroa dependens S. glandulosa (voucher under GB2125). Salpichroa S. gayi Benoist inUrubamba, 13º24’36’’S, 72º00’05.6’’W, Cuzco, Perú, in January 2012 (L.) Gaertn. (Ray and Gupta, 1994) Salpichroa (Hook.) Miers subsp. subsp. Miers (Hook.) S. didierana were collected inCorrientes, Argentina, in December 2008. A voucher specimen for taxa studied has been deposited at the Museo Botánico Córdoba (CORD), (Hook.) Miers in Pariahuanca, 11º59’28.68’’S, 74º58’18.11’’W, Junín, Perú, in S. glandulosa 50 taxa reaffirms that taxareaffirms

≥ Jaub. Jaub. Urubamba,in 13º06’51.5’’S, 72º20’50’’W, Cuzco, Perú, in January 29.97 Salpichroa μ 245 M), M), these results contribute to deeper structure–activity glandulosa (0.2 nm thick) plates. Preparative TLC separations were subps. Jaborosa species. weddellii S. leucantha S. in Urubamba, 13º17’07.8’’S, 72º02’58.6’’W, Cuzco, [ 8 ] and and (Benoist) Keel in Cercado, 17º42’09.1’’S, 1 H and 100.63 MHz for Physalis hispida Pereyra, Quip. Pereyra,inSalpo- &S.Leiva Salpichroa S. proboscideaS. S. scandensS. Salpichroa S. ramosissima S. S. comptum are distant in the systematic of S. microlobaS. 2 genus since have they also Cl Dammer (sample 1) in Tafí, (Waterf.) Cronquist S. scandensS. 2 Benoistin Paucartambo, :MeOH (8:2). Miers Miers in Matucana, 13 S. micrantha C, C, while 2D spectra (voucher under GB S. tristis S. Keel inCanta, S. tristis δ Salpichroa (sample 2) in ) downfield S. tristis Miers var. Benoist var. [ 32 ] and at at var. S. S. Accepted Article H-4 2.68 (1H, phase was with extracted CH phase was with extracted CH This by is protectedarticle reserved. Allrights copyright. pressure. The residue obtained (22.9 g) was defatted by partition in n-hexane−EtOH−H temperature immediately after collection. After 24 h. the sample was and filtered the solvent was evaporated reduced at The fresh aerial of species from Salpichroa of compounds Isolation and Extraction TLC were profiles and combined analyzed by silica silica gel 60 G CC. Elution with CH I-II (27 mg), which were purified by TLC (CH H-4 was fractionated by TLC (CH mg) was column chromatographed over silica gel 60 G, using CH (1H, (1H, = 8.2, 1.2 Hz, H-16), 6.88 (1H, 2,13,15,17-tetraen-1-one mg) was chromatographed on a gelsilica column, using AcOEt as the eluent to afford salpichrolide C (25.2 mg). resulting respectively. Fraction II (1.46was gr) separated by column chromatographywith CH chromatographed over silica gel60 using G, CH EtOH−H EtOH−H with CH the elution, to afford salpichrolide A (185.9 mg) and an impure fraction (17 mg) whichwas further purified by preparative TLC 20. Elution with MeOH afforded twenty three fractions. All fractions with similar TLC profiles were combined and analyzed by under reduced pressure. This resulting residue (6.2 g) was initially fractionated by column chromatography over Sephadex LH- Sephadexwithand elution LH-20, n-hexane under reduced pressure, with the resulting residue (1.85 g)being initially fractionated by column chromatography over a dryness at reduced pressure. The residues obtained were fractionated by column chromatography over Sephadex LH-20, using profiles were combined and analyzed by fractionated by preparative TLCwith CH to give 239.0 mg of salpichrolide A, animpure fraction II-I, and 150.0 ofmg salpichrolide Fraction C. II-I (40.0 mg) was immediately after collection. The residue obtained after evaporation theof solvent was partitioned with n-hexane C-16), 91.7 (CH, C-26), 77.5 (C, C-5), (CH,67.8 C-22), 65.2 C-24), (C, 64.0 (CH, C-6), 64.0 C-25), (C, (C,52.9 C-10), 43.3 (C (CH, C-3), 140.9 (C, C-17), 137.8 C-13),(C, 137.3 (C, C-14), 129.5 (CH, C-18), 129.1 (CH, C-2), 126.1 (CH, C-15), 125.7 (CH, (dried film) film) (dried pressure. The residue obtained (31.1 g) was defatted by partition in n-hexane−EtOH−H temperature immediately after collection. After 24 h. the sample was and filtered the solvent was evaporated reduced at gel, eluting with CH NMR to give two fractions containing withanolides (fractions andI II). Fraction (738.5I mg) was chromatographed over silica (10:3:1), the aqueous EtOH phase was concentrated, and the resulting aqueous phaseEtOH was extracted first with CH 1.31 (3H, and afterwardswith EtOAc. The CH n -hexane α β The fresh plants ofeach remaining plants The fresh The fresh aerial plants of aerialplantsThe fresh Salpichrolide V ), 2.16 (1H, ), 3.35 (1H, d , J 2 2 2

=9.9 Hz, H-26), 4.07 (1H, Cl O phase beingwashed with n-hexane (3 × 500 mL) and EtOH evaporated at reduced pressure. The resulting aqueous O phase beingwashed with n-hexane (3 × 250 mL) and EtOH evaporated at reduced pressure. The resulting aqueous fractions thathadbeing similar combinedprofiles TLC andanalyzed by s m 2 , H − ν − , H-20), 2.55 (1H, max CH MeOH, 94:06 to give salpichrolide A (3.5 mg) and salpichrolide I (7.4 mg). Fraction II (89.7 mg) was subjected to 3 -27), 1.29 (3H, 3433, 3048, 1686, 1031, 735 cm 2 m d Cl , , H-9),, 1.77 (1H, 2 J 2 (2:1:1) mixture for the elution, affordto between twenty and twenty-five fractions. All fractions with similar Cl

= Hz,9.9 OH-26), 3.14 (1H, [ (20 2 − MeOH mixturesMeOH of increasing polarity of (100:00–85:15) to yield two impure fractions (31.0 I-I mg) and ] S S. scandensS. ( ,22 1 ). ). amorphousWhite solid. [ 2 2 2 S. scandens S. R Cl dt s Cl Cl brs , H ,24S,25 , 2 2 2 − (3 x 500 Theml). CH (3 x 250 Theml). CH J 3 MeOH 98:02)yieldof salpichrolide mg andMeOH to 8.0 of9.6 compound mg A , H-18), 6.57 (1H, (1H, 6.57 H-18), , = 14.5, 2.9 Hz, H-7 -28), 1.18 (3H, 2 Cl dd 2 t , Cl 2 J from Argentina (590 g) (samplewere 1) triturated with (1000 EtOH mL) at room , − 2 = 2.9 Hz, H-6), 3.79 (1H, S J Salpichroa and EtOAc extracts were over dried anhydrous Na MeOH MeOH (100:00–85:15) afforded salpichrolide S (8.1 mg) and a mixture (26.2 mg)which 2 = 14.4, Hz, 2.5 H-23a), 1.51 (1H, ,26 1 Cl H NMR, resulting in three fractions containing withanolides (I from Bolivia 2 R − − 2 Cl MeOH (94:04), yielding mg2.8 of salpichrolide A and 3.7 mg of compound CH )-6 1 H Fractions NMR. showing doubtful 2 − β 2 −1 2 Cl MeOH 98:02) to afford 8.8 mg of salpichrolide A and 4.8 mg of salpichrolide S, Cl -chloro-22,26:24,25-diepoxy-5 brt . d species studied (ca. 1.0 kg) were triturated with EtOH at room temperature temperature room at EtOH with triturated were kg) 1.0 (ca. studied species 2 , 1 2 mixtures of increasing polarity (100:00–85:15) for the elution, with the H NMR(CDCl − ddd , J MeOH (2:1:1) affording thirty four fractions. All fractions with similar TLC J = 7.1 Hz, H Hz, 7.1 = β

2 2 = 11.8 Hz, H-8), 2.97 (1H, (1.0 kg) (sample 2) werewith triturated EtOH (2000 mL) at room ), 2.41 (1H, 2.41 ), α Cl Cl , ] J 21 2 2 extract was dried (Na extract was dried (Na = 10.2, 5.1, 2.2 Hz, H-3), 5.91 (1H, D =-3.4 ( ddd 2 3 Cl 3 -21); , 400MHz) brd c 2 , − = 0.6, CHCl = 0.6, J MeOH MeOH mixtures of increasing polarity (100:00–90:10) for , = 11.3, 5.7, 2.6 Hz, H-22), 3.41 (1H, J 13 = 12.2 Hz, H-11 C NMR (CDCl C NMR m , H-23b),, 1.36 (3H, δ α 7.14 (1H, 2 2 ,26-dihydroxy-17(13 1 SO SO 3 H NMR again. again. H NMR m ). UV (MeOH) (MeOH) UV ). 1 H NMR spectra were column were column H NMR spectra , H-12 2 4 4 Cl ), filtered, and evaporated dryness to ), filtered, and evaporated dryness to 2 2 2 3 − O (10:3:1),with the resultant O (10:3:1),with the resultant , 100 MHz) MeOH mixtures of increasing polarity α α d ), 2.23), (1H, 2 ), 2.75 (1H, (1H, 2.75 ), SO , J = 8.2 Hz, H-15), 6.94 (1H, (1H, 6.94 H-15), Hz, =8.2 dd 4 , filtered,and evaporated, to s λ , , H max J = 10.2, 2.8 Hz, H-2), 4.92 δ 3 -19), 1.33 (1H, (log 203.0 C-1), (C, 141.1 → m − brd 18) abeo-5 III).Fraction I(316.1 , H-7 ε ) 216 (4.20) nm. IR , 1 J . Fraction. III (340.7 dt = 17.0 Hz, H-12 α , ), 2.22 (1H, J − = 19.8, 2.8Hz, EtOH α m -ergosta- , H-11 −

H 1 2 Cl dd . 2 O m 2 , β

, 1 J ), β H H, H,

), ), Accepted Article manuscript. V.E.N. and G.E.B. have equal contribution. Both participated to writing, and all the authors read and approved the final the species studied in work;this V.E.N. participated in designthe and coordination of the study. performed biological determinations; V.P.C., P.A.S., and J.C.C. interpreted biological data; G.E.B. provided and identified all This by is protectedarticle reserved. Allrights copyright. the field trips in Peru and identified some A.V.B. carried out the phytochemical analysis and participated in field trips; S.L.G. organized and participated in the majorit Statement Contribution Author assistance, M. Gritti for the design of Figure 1, and Dr. Paul Hobson, native speaker, for revision of this manuscript. CONICET and MINCyT(Argentina) for a fellowship. are gratefulWe to G. Bonetto for NMR assistance, J. Cedrón for the logistic Cooperación Científico-Tecnológica MINCyT-Argentina and CONCYTEC-Perú 13/02. (PE 2014-2016). A.V.B. thanks workThis was supported bygrants from CONICET (PIP Acknowledgements Supporting information for this ar Material Supplementary Treatment of cancer cells. evaluation Biological maintained at 37 ºC inahumidified withincubator a 5% CO LNCaP, PC3, MCF-7 and T47Dwere cells obtained from the American Type Culture Collection (Manassas, VA, USA) and and culture reagent Cell C-20), 38.8 (CH, C-9), 37.1 (CH Aldrich Chemical Co. (St. Louis, MO, USA). Diego, CA, USA). The data were recorded as means ± standard deviations from triplicates. Statistical Analysis reader. The IC and were cells incubated at 37 ºC for 1h. Absorbancewas detected 490at nm ona Thermo Scientific Multiskan FC plate formazan-colored product (A 490 Followingnm). the manufacturer’s instructions, 20of µl MTS reagent were added to each well Proliferation Assay System (Promega Corporation, Madison, USA), in which cellsviable convert MTS tetrazolium into a Citotoxicity assay prepared eachfor individual dose. withfreeserum medium and wells withan amount equal DMSO of were used negative as controls. Triplicate wellswere of each compound and incubated for 48 h. In parallel, cells were withtreated doxorubicin as standard anticancer drug. Wells incubation for 24 h, the media were aspirated and replaced with 100of µl serum free medium containing different concentration (Invitrogen). MicroVet, Buenos Aires, Argentina) supplemented with 10% fetal bovine serum (Natocor, Córdoba, Argentina) and antibiotics C cells/100 PC3, µl), MCF-7andT47D (5x10 cells 19.1 (CH 28 H 35 ClO 3 , C-28), 17.6 (CH 17.6 C-28), , 5 Na, 509.2065).Na, 33 Doxorubicin was kindly provided as a gift by Dr. Gonzalo Sequeira (IBYME) and was purchased from Sigma- 50 value wasdefined concentrationas value the of compound50%yielding cell survival. . MTS cell viability analysis was performed 48 h post treatment using the Cell Titer 96 AQueous One Solution . Statistical.was Analysisconducted 5.0Padusing Graph the Prism software Pad(Graph Software San Inc., 3 Optimumseedingdensity for 96-well was determinedcell plates both for (10cells lines.LNCaP , C-21), 16.9 (CH 2 , C-4), 35.0 (CH 35.0 , C-4), ticle is available on the WWW under h

Salpichroa 3 , C-27), 16.3 (CH 2 , C-7), 34.0 (CH 3 cells/100 µl) were seeded into each well of a 96-well microtiter plate. After species;designed V.P.C andP.A.S. biological experiments; P.A.S. 112, 2013-2016),112, SECyT 2 /95% air atmosphere inRPMI 1640 medium (EMEVE Medios, Lab 3 , C-19). HRESITOFMS HRESITOFMS , C-19). 2 , C-23), 32.9 (CH, C-8), 31.0 (CH ttp://dx.doi.org/10.1002/MS-number. -UNC (Res. 366, 2016--UNC (Res. m/z [M+Na] + 2 509.2066 (calcd for , C-12), 26.0 (CH 1017), Programa de 2 , C-11), y of y of

4

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re-Cytotoxic illeul, teroids’, teroids’, Physalis