September 1968
SAXGIVAMITCIN
stirred on a steam bath for 5 hr. Tlc analysis of the solution showed the presence of four major orange-yellow spots: Rf 0.23 (11), Rf 0.3.3 (111), Rr 0.52 (I), and Rf 0.62 (I\.) in the approximate ratio 2: 1 : 3.5: l.:, besides other colored by-products a t Rf .cin. Titration of its hydrochloride shows two regions of equilibrium (pka = 2.9 arid 5.6) (E'igure 1B). This as well a s the ir spectral peak at 5.80 I.( suggest that deswmidosangivam~cinhas a car1 group. It can be esterified to a moriometh;\,l , C,:IH,6S.10,,which s h o w a sharp peak :It 5 . S ; p . It :dso has 1: methoxyl peal; at r 6.00. ig discussion it is evident that entose residue. an aromatic amino mide group. It has tn-o ( 7 1.42 and 1.53) as part of' the het,erocyclic Of the many possibilities, a p>-rrolo[2.3-tZ]pyr skeleton presetit in other uctinomj-cete metabolites such : ~ s tuhercidiii" :uid toyocamyciri4 appeared probable. comparison of the uv spectra of eangivamycin :iud t o>.ocanij*cinshon-ed that they :ire very similar iricluding the iipect,rzil shift in acid medium. Their rcspective elemeritary compositiciris suggested that s:irigiv:tni>.cin :ind toyocamyciri may have the same carbon skeleton with the former having a carboxanlick while the latter has a nitrile group. That the :hove hypothe is correct is shown in +cveral \\ays. E'iwt, wllraline hydrol>,sis of toyocam>.c 4 i i yields xi iicitl which is identical n-ith desamidos:irigivam>.cin. Scxt t hc tctraacetyl deriv:ztive of s:ingiv:rniyciii c111 be dehydrated by means of POCI, to the tetraacetyl derivutive of toj,ocamycin. The acetates were compared in the forni of their crystalline picrate salts. In the ir both compounds show the characteristic sharp peak at 2230 cm-' ( C S ) . Finally. hy selective acid hydrolysis of toyocamycin in 2 N HC'I at 100"; sangivamyciri itself is isolated from the
.4c.oHoH2cfj HO
3 I
bCOCH,
v
OH
IV
NHCOCH,
CH,COb
4C 0
NHCOCH,
I
CH.,COO OCOCH3
VI
111 vien- id' tlie $tructural similarit!- het\veeri tubercidiit (11). to).ocani>-ciii (III), : i d sangivamycin ( I ) it is of ititcrest to rmmirie their bio1ogic:il activities;. A%lthough311 t h r w exhibit i: high degree of cytotosicit!. towurd Hc1,t-l cells I'ii c i f i v , only saiigivamycin s h o w significarit Ztctivity against leulremiu L1210 iii mico. Because of this. :t brief stud). w~ismade of the available derivatives against 1,1210 iii mice. The structur:tl chaiiges cover three areas: the amide group, the aniiiio group. :tncl the pelit Hydrolysis of xi11 t o tlesaniido~aiigivutii>.ciri ill-) leads t o loss of activity. 1~'orrnation 01 :I er iwtores 2ictivitj. slightly. 1iestor:itioii (of activity is much higher it1 compourds with riitrogeti substitution. 1;or itistarice. the S-niethylmiide, the hydroxamic acid. aiid t,he hydrazide of clesamidosaligivamycin all show significaiit activity. L%niongthose, the hydrazide appears to be the most active. Deamiiiatioit of s:iiigivamyciIi t o the oxydeaainitio derivative le:& t o loss of activity. Also, rtcetylatioii of t'he amiiio group iis well :IS of the h>droxyl groups of the pentose britigs nbout iriactivation. 1,ilcewise. the periodate-osidatit,n product is also iriactivo. Table I sliou-> thc ;ipproxiniate dose ranges aiitl t 1i(. corrwponding i i i ( w : i , < p i r r s;lii.viv:il times ohtairied ivitli s
September 1968
SANGIVAMTCIN
Compound
Sangivamycin
Ilexamido JIethyl eater of deaamido
K-Methyl
Iiydrosarnic :wid of deb rniido
Osydeuamino Tet raacet yl Periodate-osidatioii product
Dose, mdkg
0.6 0.4 0.27 20 3 2 1 .3:5 0.88 2.4 1.8 1.2 1.0 6 4 2.7 2 . .j 2 .0 1 .3 1.0 20 20 20
Extension of survival time, 9%
1.50 160 1:53
98 132 132 139 138 149 14.5 144 128 144 127 119
134 162 149 14 1 102 100 98
some of these derivatives in leukemia 1,1210 carried out according to the protocols of the Cancer Chemotherapy Satiorial Service Center. A compound is considered active if the survival time is l%y0or higher of that of untreated animals.
Experimental Section Mclting points are corrected and were determined using a Fisher-Johns apparatts. Sangivamycin war; recovered from the broth by adsorption on Uarco (2-60 (1.5%) followed by several elutions with 0.05 .V hIeOH-HC1. The combined eluates were concentrated without neutralization and set aside in the refrigerator. The crude crystalline solid of sangiva cin hydrochloride which separated out was filtered and recr allized from hot H,O. I t separated as c )lorless long needles, mp 250-252'. Anal. Calcd for CI2H,,N50,.HCl-T-r,0: C, 39.75; H, 5.00; N, 19.30; C1, 9.78. Found: C, 40.14; H, 5.30; S , 19.26; C1, 9.94. Tetraac ety1sangivamycin.-A mixture of sangivamycin ( 1 g ) j AclO (10 ml), and pyridine ( 2 ml) was heated at 100° for 2 hr. The eo ,led solution was diluted (HgO) and extracted (CFICI,) after 30 min. Th? solvent extract was washed (NaHC03-H20) and concent'rated to dryness. The acetate crystallized from EtOH 2s colorless p isms, mp 153-155'. .Anal. Calcd for CJI,sX&.0.5H;O: C, 49.39; H, 4.93; X, 14.41; acetyl, 35.40. Forind: C, 49.06; H, 5.21; Y, 14.39; acetyl, 34.58. 0xydesaminosangivamycin.-A s o h tion of sangivamycin (0.3 g) in AcOH (30 ml) was cooled to 5' and treated with NaN02 ( 1 g in 10 ml). After 30 min at .io, the blue solution was heated at 80' for 30 min and then concentrated to dryness. The residue was crystallized from lIeOH-H20. The product crystallized as colorless needles, mp 290-292'. Anal. Calcd for C12H14S4Ofi: C, 46.45; H, 4.55; K, 18.06. Foiind: C, 46.25; H, 4.68; N, 18.07. Desamid0sangivamycin.-Sangivamycin ( 2 g) was boiled under reflux with 2 iV NaOH (200 ml) for 3 hr. The cooled solution was diluted (H,O) and passed through a column of Amberlite IK-C5O in H + form. The effluent. and wash were concentrated to dryness and the residue was crystallized as a hydrochloride from MeOH. The hydrochloride crystallized as colorless long rectangular plates, mp 236-238'. Anal. Calcd for C12H14N406.
94 1
HCl: C, 41.50; H, 4.34; N, 16.12; C1, 10.15. Found: C, 41.00; H, 4.52; 12,15.89: C1, 10.18. Desamidosangivamycin Methyl Ester.-Desamidosangivamycin ( 1 g) was boiled under reflux with l0yo &So4 in MeOH (100 ml) for about 24 hr. The cooled solution was diluted with water (100 ml) and passed through a column of Dowex-1 in acet,ate form. The effluent and wash were concentrated to dryness and the solid crystallized from MeOH. The ester separated as colorless prisms, mp 216-218". Anal. Calcd for C I ~ H I ~ N ~ O ~ ~ : C, 48.15: H, 4.97: N, 17.28; OCH3 ( l ) , 9.60. Found: C, 48.10; H, 5.07; N, 17.11; OCH,, 9.69. Periodate Oxidation of Sangivamycin and Hydrolysis.Sangivamycin ( 1 g) was dissolved in 1 HC1 (30 ml) and treated with 0.2 J I aqueous H J 0 6 (30 ml). rlfter 20 hr the crystalline solid was separated, dissolved in H,O (20 ml), and treated with a slight excess of NaHCO,. The crystalline base was separated and recrystallized from H20. The product was a colorleds crystalline solid, mp 170" dec. Anal. Calcd for CI,H1J;O>.3H,O: C, 39.89; H, 5 . 3 0 ; N, 19.36. Found: C, 39.79: H, 5.15; S , 19.39. A portion of the oxidation product (0.5 g) was boiled under reflnx with 6 S HC1 for 6 hr. The dark colored mixture was concentrated to dryness and the residrie was crystallized twice from AIeOH. The product appeared as colorless needles, mp 285289". Anal. Calcd for CiHcNjO,.HCl: C, 39.17; H, 3.58: N, 26.00. Found: C, 38.80: H, 3.90; irj, 25.54. Conversion of Tetraacetylsangivamycin to Tetraacetyltoyocamycin.-A solution of tetraacetylsangivamycin (0.2 g) in CHC1, ( 2 5 ml) was boiled iinder reflux with POC1, (0.3 ml) for 3 hr. The mixtnre was concentrated to a small volume and diluted wit:> HgO. After 30 min it was neutralized and extracted (CHCI,). The extract wa.3 concentrated and the residue was converted to a picrate, which crystallized from llenCO-l\IeOH as yellow long needles, mp 160-162". Mixture melting point with the picrate of tetraacetyltoyocamycin was undepressed. Conversion of Toyocamycin into Sangivamycin.--A solutiun of toyocamycin (0.2 g) in 2 S HC1 ( 5 ml) was heated at 100" for aborit 4 hr. I t was cooled in an ice bath and the solid whirh separated out was filtered and crystallized from AIeOH. It separated as colorless long needles, mp 250-252", alone or in admixture with sangivamycin hydrochloride. N-Methy1sangivamycin.-To a suspension of desamidosangivamycin methyl ester (0.n g) in NeOH (15 ml) was added 4 0 7 aqueous MeXH2 (10 ml). The mixture was warmed to obtain a clear solution and let stand at room temperature for 24 hr. The solution was concentrated to dryness and the stlid crystallized as its hydrochloride from hIeOH. TEe compound is a colorless crystalline d i d , mp 242-246". Anal. Calcd for C I ~ H I ~ ~ - ~ O : . HC, C I :42.80; H, 5.48: K, 19.20; C1, 9.70. Foiind: C, 42.76; H, 5.24; N, 19.11: C1, 9.29. Hydroxamic Acid of Desamidosangivamycin.-A mixture of sangivamycin ( 1 g), HO?;H3+C1- ( 1 g), and pyridine (10 ml) was boiled under reflux for 6 hr. The cooled mixtiire was concentrated t o dryness. I t was then dissolved (HZO) and passed through a colrinin of Dowex-1 in acetate form (50 ml of the resin). After washing (H20), elution was carried out with 2 The eluate was concentrated to dryness and the solid c from MeOH as its hydrochloride. The compound is a colorless crystalline solid, mp 216-218". I t gives a blue color with FeC13. And. Calcd for C1,H,,N,OG. HC1: C, 40.00; H, 4.44; C1, 9.i8. Found: C, 40.19; H, 4.59: C1, 9.29. Desamidosangivamycin Hydrazide.--.& mixture of desamidosangivamycin methyl ester (0.5 g), NeOH ( 5 ml), and hydrazine ( 2 ml) was warmed until a clear solution was obtained. rlfter 24 hr a t room temperature, ether (25 ml) was added and the solid was filtered o f f . I t was recrystallized from AIeOH-H20. The compound is a colorless crystalline solid, mp 238-240". ilna/. Calcd for C12H16N60;: C, 44.44; H, 4.97; N, 25.92. Found: C, 44.29; H, 5.22; N, 25.56. Alternatively, a mixtlire of sangivamycin (1 g), n-BitOH (25 ml), and hydrazine (4 ml) was boiled under reflux for 20 hr. The solution was cooled and diluted (EtZO), and the solid was filtered. It. was crystallized from i\IeOH-H,O. Paper chromatography and the ir epectrum of the sample are identical with those of t,he sample described above.
