AU603418B2 - Sparsomycin (sc-rs) compounds having antitumor activity, a process for their preparation and pharmaceutical compositions containing sparsomycin (sc-rs) compounds - Google Patents

Description

I

I-ff-- I OF AUSTRALIA COMMONWEALTH OF AUSTRALIA Patent- Act 1952 COMPLETE SPECIFICATION

(ORIGINAL)

Application Number Lodged Complete Specification Lodged Accepted Published U~ f Priority Related Art Name of Applicant Address of Applicant Actual Inventor/z Address for Service STICUTING KATHOLIEKE

UNIVERSITEIT

Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Henricus Carl Joseph Ottenheijm F.B. RICE CO.

Patent Attorneys 28A Montague Street, Balmain N.S.W. 2041 Complete Specification for the invention entitled: Sparsomycin (Sc Rs) compounds having antitumor activity, a process for their preparation and pharmaceutical compositions containing sparsomycin (Sc-Rs) compounds The following statement is a full description of this invention including the best method of performing it known to us/A:- -1la- The invention relates to novel sparsorrycin compounds having valuable pharmacological properties, in particular a strong antitumnor activity, a relatively low toxicity, a good solubility in aqueous media and a potentiating effect on the cytostatic activity of other cytostatics, such as, for example, cis-platinumn and The invention also relates to pharmaceutical compositions containing such novel sparsomycin (Sc-R 5 compounds, and to a process for preparing said novel 0 sparsomycin (Sc-Rs) compounds.

Sparsomycin is a metabolite of Streptomyces sparsogenes (Antimicrob. Agents Chemother. (1962), 780) and of Stepomyes cuspidosporus (Chem.Abstr. (1l 67), 66, 54328) and has the structure formula: 00,O 0 H

CHC

3 U3 Sparsomycin has attracted mtucht attention on accounc. of its interesting biological activity. This activtity is primarily a result of a strong inhibition of the protein biosynthesis resulting in a decline of the protein synthesis and concomitant biological effects.

It has been shown (Ann.Rev.Microb. (1971), 25, 488;* FEBS Lei~t (1974), 40, S63; Molec.Bical.Biochem.Biophys.

(1979), 30) that the site of interaction oe sparsomycitn I a u

I.

i a.

a i a aa" I is in the large ribosomal subunit, where it prevents peptide transfer by interfering with the peptidyl transferase centre. The action of sparsomycin has been demonstrated in prokaryotic cells Slechta: "Antibiotics Editors: 5 D. Gottlieb and P.S. Shaw, Springer Verlag, New York (1967), 4-0; Can.J.Microb. (1967), 62, 595), eukaryotic cells (Biochem.Biophys.Res.Comm. (1966), 23, 453; J.Antibiot.

(1978), 31, 598; Biochem. (1977), 16, 3209), including transformed cells (J.Med.Chem. (1977), 20, 337; Antimicrob.Ag.

Chemother. (1962), 772; Biochim.Biophys.Acta (1979), 563, 479; Cancer,Res. (1972), 32, 398) and virus-infected cells Virol. (1979), 29, 114; J. Gen. Virol. (1968), 2, 143), and in various cell-free systems (Biochim.Biophys.Acta (1966), 129, 642; Biochim.Biophys.Acta (1976), 447, 460; Proc.Natl.Acad.Sci. U.S.A. (1968), 61, 726; FEBS Lett. (1975), 52, 236). On the other hand, sparsomycin is not active against whole reticulocytes (Biochim.Biophys.Acta (1966), 119, 109), which is attributed to sparsomycin's being unable to penetrate these cells. The behaviour of sparsomycin with regard to its inhibitory action and its influence on the polyribosomes has also been investigated in vivo (Proc.Natl.Acad.Sci. U.S,A. (1968), 59, 854, J.Natl.Canc.Inst. (1979), 63, 81; Biochem.Pharmacol.

(1974), 23, 857).

In connection with the demonstrated activity of sparsomycin against transformed cells and various tumOrs (Antimicrob. Agents Chemother. (1962), 772), it has been investigated as a potential cytostatic compound, but clinical tests (CancerChemother.Rep. (1969), 43, 29) revealed eye toxicity.

In order to gain an insight into the biochemical interaction mechanism and the relationship between structure and activity, various researchers have prepared derivatives of sparsomycin and investigated their activity. In the absence of a total synthesis, however, only a limited I_ 1 *u -ii -3number of derivatives could be investigated, which each differed from sparsomycin itself in various structural parameters. As a result, only limited conclusions could be draw- as to the role of the various structural fragments- Another problem in the interpretation and comparison of the available information as to the relationship between structure and activity in sparsomycin is that the biological activity of the various derivatives has been determined in different systems: in vitro in a KB cell culture (J.Med,Chem. (1977), 20, 337) or in cell-free ribosomal systems (Biochem.Biophys.Res.Comm.

(1977), 75, 563; J.Med.Chem. (1978), 21, 177), in vivo in the P-388 system and the Walker 256 system (J.Pharm.Sci.

(1975), 64, 825).

Recently a few total syntheses for sparsomycin an derivatives thereof have become available (J.Am.Chem.Soc.

(1979), 101, 1057; J.Org.Chem. (1981), 46, 3273; J.Org.Chem.

(1981), 46, 5408). These total syntheses enabled the present inventors to carry out an investigation in which various suitably selected derivatives of sparsomycin were prepared and their cytostatic activity was investigated in an in vitro clonogenic L 1210 assay, whereby the inhibition of colony formation was measured.

Said prior investigation, which forms the basis of Dutch patent application No. 8204224 (US patent No.

4,581,360) concerned sparsomycin (Sc-Rs) derivatives in which the S-bound termin .l methyl group was replaced by a more lipophilic group, preferably a C 2

-C

2 0 alkyl group, aryl group, aralkyl group or alkaryl group. Exemplified representatives of said lipophilic group are n-octyl and benzyl. Thhe n-octyisparsomycin is poorly dissolved in water (about 0.2 mg/ml water) whereas benzylsparsoiycin dissolves much better in water (1.5 mg/ml water).

The investigation concerned further comprised sparsomycin (S 0 -Rs) derivatives in which the hydroxymethyl group was etherified or esterified.

The compounds concerned were prepared by a process which started from D-cystelne or D-cystine.

It has now been found that novel sparsomycin (Sc-Rs) compounds can be obtained from much cheaper L-amino acids as starting material, said novel compounds being characterized by a different group at the location of the hydroxymethyl group and having valuable pharmacological properties.

In a first aspect, the present invention provides a sparsomycin (Sc-Rs) compound having the general formula 0 0 0 N,

S

R

H

wherein: RI represents to., C 1

-C

6 alkyl or substituted C 1

-C

6 alkyl, with the exception of hydroxymethyl, alkoxymethyl and acyloxymethy].; phenyl or substituted phenyl; heterocyclyl or substituted heterocyclyl; benzyl or substituted benzyl; heterocyclylmethyl or substituted heterocyclylmethyl;

C

3

-C

7 cycloalkyl or substituted C 3

-C

7 cycloalkyl;

C

3

-C

7 cycloalkylmethyl or substituted C 3

-C

7 cycloalkyln~ethyl;

R

2 represents Cl-C 1 0 alkyl or substituted C 1 -Cl 0 alkyl; phenyl or substituted phenyl; heterocyclyl. or substituted heterocyclyl; benzyl or substituted benzyl; heterocyclylmethyl or substituted heterocyclylmethyl;

C

3

-C

7 cycloalkyl or substituted C 3

-C

7 cycloalkyl;0 iili-

C

3

-C

7 cycloalkylmethyl or substitUted

C

3

-C

7 cycloalkylmethyl;

R

3 represents hydrogen; or R3 and R 1 combined with the nitrogen atom and carbon atom to which they are respectively attached, form a three to seven membered, substituted or unsubstituted heterocyclic ring which may contain up to 3 additional heteroatoms in the ring selected from the group consisting of N, 0 and S; and pharmaceutically acceptable salts thereof.

In a preferred embodiment of the invention, R1 represents

C

1

-C

4 alkyl, optionally substituted by one or more substituents selected from the group consisting of S03H, COOK,

CONH

2

CONHR

4

CONR

4

R

5

NH

2

NHR

4

NR

4

R

5

NH-C(NH

2

)=NH,

NHCOR

4 Nj3, N0 2 thiol, thioether, imidazolyl, and halogen; phenyl or benzyl, optionally substituted by one or more substituents selected from the group consisting of OH, SH, S03H, C)OH, C 1

-C

4 alkyl, C 1

-C

4 alkoxy, Cl-C 4 alkylthio,

C

1

-C

4 acyl, C 1

-C

4 acyloxy, C 1

-C

4 acylamido, N3, N0 2

SNH

2

NHR

4

NR

4 R5, 0-sulphate, 0-phosphate, and halogen; imidazolemethvl or 3-indolylmethyl;

R

2 represents C,-Cg alkyl optionally substituted by one or more substituents selcted from the group consisting of OH, SH, S03 25 4 COOH, COGR, CONH 2

CONHR

4

CONR

4

R

5

NH

2

NHR

4

NR

4

R,

NHCOR

4 N3, N02, and halogen, phenyl or benzvl, optionally substituted by one ur more subztituents selected from the group consisting of tI, SH, 4 So 3 H, Co00H COOR 4, C-c 4 lkyl, C-C 4 alkoxy, C 1

-C

4 alkylthio,

CI-C

4 acyl4 C-C 4 acyloxy, Cj-C 4 acylamido, N3, N02,

NH

2

NHR

4 t Nk 4 k, and halogen;

R

3 represents hydrogen; or R 3 and Rl, taken together, represents -(CH 2

R

4 represents C 1

-C

4 alkvl, phenyl or benzyl;

R

5 represents C 1

-C

4 aiylt -6and pharmaceutically acceptable salts thereof.

Specific examples of novel sparsomycin CSc-Rs) compounds according to the present invention include the compounds: 5(2a): ala-sparsornycin (Rl=methyl; R 2 =Methyl; R 3 =hydrogen; prepared from L-alanine as starting material); val-sparsomycin (Rl~isopropyl; R 2 =methyl; R 3 =hydrogen; pre pared from L-valine as starting material); ile-sparsomycin (Rl~sec.butyl; R 2 =methyl; R 3 =hydrogen; prepared from L-isoleucine as starting material); (2d) phe-sparsomycin (R'benzyl; R 2 =methyl; R 3 =hydrogen; prepared from L-phenylalanile as starting material); (2e) n-butyl-ala-sparsomycin (Rl=methyl; R 2 =n-butyl;

R

3 =hydrogen); n-octyl-ala-spa.rsomycin (Rl~methyl; R 2 =n-octyl;

R

3 =hydrogen); (2g) pro-sparsomycin having the structural formula 0

\H

H 3 0 C3

H

1* preparpd from T-proline as starting material),; ethyl-ala-sparsomycin (R 1=methyl; R 2=ethyl; R 3=hydrogen).

Apart from the significant economic advantage that the novel sparsomycin (Sc-Rs) compounds can be prepared from much cheaper L-aminoacids as starting material, they appear to combine a strong antitumor activity with A good solubility in aqueous systems and a relatively low toxicity.

As has been described earlier, the antitumor activity is correlated to the lipophilicity of the compound.

Si -7- There appears to be an optimum in lipophilicity of sparsomycin analogues since compound with five carbon atoms in the R, alkyl side chain (n-pentyl-sparsomycin) shows maximal in vitro and in vivo activity.

Of previously examined compounds n-octyl-sparsomycin seems to be beyond such an optimum. Additionally, this coiapound is very difficult to handle because of poor water solubility. The high LD50 values of n-octyl-sparsomycin depends on extensive plasma protein binding and rapid total body i 10 clearance of the drug.

The compounds of the present invention, such as, for example, ala-sparsomycin (2a) and in particular ethyl-alasparsomycin (2h) surprisingly appear to be closer to the optimum in that they combine a higher water solubility with a stronger antitumor activity and a relatively low toxicity.

The best combination of pharmacological properties is expected to be found with sparsomycin (Sc-R s compounds according to the present invention, in which R 1 contains one or more substituents that improve the water solubility, such as, for example, SO 3 H groups, and in which R 2 represents an alkyl group having about 2 or 3 to 4 or 5 carbon atoms or a benzyl group.

The present invention also provides pharmaceutical i compositions comprising a pharmaceutically acceptable carrier, dilutent, solvent or excipiens and a therapeutically effective amount of a sparsomycin (Sc-R s compound according i to the invention.

The compositions may also contain other active ingredients, such as other antitumor agents and may be made up in any pharmaceutical form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenteral administration such as sterile solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions LL l J a" -8which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.

Optimal dosages and regimens of active ingredient for a given mammalian host can be readily ascertained by those skilled in the art. It will, of course, be appreciated that the actual dose used will vary according to the particular composition formulated, the mode of application and the particular situs, host and condition being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the condition.

The novel compounds according to the invention appear very useful in combination therapies with known cytostatics such as cis-platinum and which have wide-spread clinical use, Even very low nontoxic doses of the novel compounds result in a significant potentiation of the antitumor activity of said known cytostatics.

In another aspect, the present invention provides a process for preparing the novel sparsomycin (Sc-Rs) compounds comprising series of reactions to convert an L-amino acid having the general formula

R

into a compound having the general formula -9- RL 0 H N S\ 2 13 and reacting said compound (12) with 6-methyl-uracil acrylic acid having the structural formu, 1 0 0 N7 oH (14) S CH3

H

More specifically, in a preferred embodiment said process comprises the steps of reacting an L-aminoacid methyl ester with di-tert.butyl pyrocarbonate to obtain an N-(tert.butyloxycarbonyl)-L-aminoacid methyl ester having the general formula

H

N COXe 4 reacting said compound with a reducing agent to obtain a compound having the general Eormula tosylating said compound to obtain a compound having the general formula BOC Oo R 3 reacting said compound with cesium carbonate and thioacetic acid to obtain a thioester having the general formula

BQC

reacting said thioester with acetic anhydride and chlorine to obtain the corresponding sulfinyl chloride, reacting said sulfinyl chloride with diazoietha.ne to obtaln the diasteroMeric c(-chloro $ulfoxides 0 BOC S C1 (8) 3? Rt I 0 -11separating said diastereomers and isolating the (Sc-Rs) a-chloro sulfoxide reacting same with a mercaptide having the general formula MSR 2 wherein M representis alkalimetal, to obtain a compound having the general formula 0

BOG

"IS)

deprotecting the arninQ group byq treatment with if~raei Acid to obtain a ompovnd having the general formnula and reacting same with 6"Methyl-uracilt acrylic~ acic1, Said process is shown schematic~ally in the followinq reaotion scheme: f-1 2

R

H

H

oc,

OH

ca Xe 3 2 3 R

R

R L

H,

BOC OTos

'N

3 RI 0 Boq,>cNS" BOC c 13R 3

R

0 a 1 0 Boc

R

3 012

R

3

R

14, H R1 t' S *1 i -13- /j The pharmacological perties and preparation i of sparsomvr-n (Sc-Rs) compounds according to the invention are shown in the following ex "rimental part, which is provided for illustrative purposes only and should not be construed as limiting the scope of the invention.

Experimental Section Melting points were determined in a Reichert hot stage and are uncorrected. 1 H NMR spectra were recorded on a Bruker WH-90 spectrometer with Me 4 Si or Me 3 SiCD 2

CD

2

CO

2 Na as an internal standard. Mass spectra were recorded on a double-focussing VG 7070E mass spectrometer. Circular dichroism (CD) spectra were recorded with an Auto-dichrograph Mark V apparatus (Jobin Yvon). For determination of Sthe specific rotation, a Perkin-Elmer 241 polarimeter was used. Thin-layer chromatography (TLC) was carried out by using Merck precoated silicagel F-254 plates (thickness 0.25 mm). Spots were visualized with a UV lamp, ninhydrin and C1 2 -TDM. For column chromatography, Merck silicagel H (type 60) was used.

References S 1. Ottenheijm et al, J.Org.Chem. 46, 3273-3283 (1981).

2. Liskamp et al, J.Med.Chem. 27, 301-306 (1984).

3. Van den Broek et al, J.Med.Chem. 30, 325-333 (1987).

N-(tert.-butyloxycarbonyl)-L-alanine methyl ester (4a) This compound was prepared in 93% yield from the hydrochloride of L-alanine methyl ester (13.2 g, 103.4 mmol) by tree-ment with di-tert,.-butyl pyrocarbonate following the procedure that has been described earlier 1 2 TLC Rf 0.80 (eluent MeOH/CHC13, 3/97). IH NMR (CDC1 3 j -14- 1.36 Cd, J=5 Hz, 3H, CHCH 3 1.50 Cs, 9H, t-Bu), 3.76 Cs, 3H, CO 2

CH

3 40.03-4,65 (in, 1H, CHCH 3 5.50 (br d, 1H, NH).

N-(tert.-butyloxycarbonyl)-L-valine methyl ester (4b) This compound was preparzed in quantitat,.ve yield from the hydrochloride of L-valine methyl este,' C25.2 g, u.15 mol) by treatmnent with di-tert,-butyl pyrocarbonate following the procedure that has been described earlier 1 2 TLC Rf 0.85 Celuent MeOH/CHCl 3 3/97). 1 H NMR CCDCl 3 '0.87 and 0.98 Cdd, J=2 Hz, 6H, CH 3

CHCH

3 1.45 (s, 9H, t-Bu), 1.77-2.37 Cm, 1H, CH 3 CHCHi 3 3.75 3H,

CO

2

CH

3 4.01-4.37 (in, IH,CHCO 2

CH

3 4.90-5.29 Cm, lH,

NH).

N-Ctert. -butyloxycarbonyl) -L-isoleucine methyl ester (4c) This compound was prepared in 99% yield from the hydrochloride of L'-isoleucine meth-l ester (27.3 g, 0.15 by treatment with di-tert.-butyl pyrooarbonate folowig the procedure that has been described earlier 1 2 TLC Rf 0,85 Celuent MeOH/CHCl 3 3/97) 1 H NNMR (CoDC 3 0 0,7 3 99 Cm, 6 H, C H 2 CH 3 CHKCH 3 1 .0 6-1. 4 1 Cm 2 H

CH

2

CH

3 1.44 Cs, 9H, t-BU), 3.27-3,67 Cm, 1HO CHCH3)t 3,73 Cs, 3H, C0 2

CH

3 4.03-4.40 Cm, lH, CHGO 2 C~i 3 4,77-5. j Cm,1H, NH).

N-(tert.-butyloxycarbonyl)-L-phenylalanine methyl ester (4d) This compound was prepared in 97% yield from the hydrochloride of L-phenylalanine methyl ester C32.4 g, 0.15 mol) by treatment with di-tert.-butyl pyrocarbonate following the procedure that has been described earlier 1 2 TLC Rf 0.85 Celuent MeOH/CHCI 3 3/97). 1 H NNIR CCDCI 3 '5 1.40 9H, t-Bu), 3.04 and 3.11 Cd, A'B part of ABX spectrum, 2Hl, CHCHi 2 3.70 3H, CO 2

CH-

3 4.36-4.73

I

I I I m, 1H, CHCO 2

CH

3 4.75--5-10 (in, lH, NH), 7.04-7.34 m, 5H, Ph).

N-(tert.-butyloxycarbonyl)-L-prolile methyl ester (4g) This compound was prepared in quantitative yield from the hydrochloride of L-proline methyl ester (24.9 g, 0.15 mol) by treatment with di-tert.-butyl pyrocarbonate following the procedure that has been described earlier 1 2 TLC Rf 0.83 (eluent MeOH,CHCl 3 1 H NMP. (CDCl 3 '5 1.44 9H, t-Bu), 1.82-2.37 (mn, 4H, CH 2

C-

2

CH),

3.33-3.65 (mn, 2H, CH 2 3.73 3H, CO 2

CH

3 4.04-4.45 (mn, 1H, CHCO 2

CH

3 N-(tert.-butyloxycarbonyl)-L-alaninol This compound was prepared in 90% yield from the estcer 4a (2.44 g, 12 iniol) by reduction with lithiumi borohydride in dry DME analogous to the method described earlie.t 1 2 The product was used for the next reaction withoit further purification. IH NMR (CDC1 3 '5 1.14 J=6.6 Hz, 3H, Cl-Id 3 1.44 9H, t-Bu) 2.71 (hr s, lH, CH 2 OH), 3.45 and 3.55 (AB part of AEX spectrum, 3.47-3.95 (in, 1H, OUCH 3 4.68 (br d, lH, NH). Anal.

Calcd. for C 8

H

17 N0 3 C, 54.84; H, 9.78; N, 7.99. Found:.

C, 54.83; H, 9.96; N, 7.96.

N- (tert. -butyloxycarbonyl )-L-valinol This compound was prepared in 81%. yield from the ester 4b (9.28 g, 40 inmol) by reduction with lithium borohydride in dry IME following the procedure that has been described earlier 1 2 TLC Rf 0.47 (eluent MeOI-l/CHCl 3 7/93). 1 H NMR (ODC1 3 '5 0.91 and 0.98 (dd, J=Z Hz, 6%-I

CH

3

CHCH

3 1.44 9H, t-BU), 1.45-1.97 (in, LU, CH 3

CHCH

3 2.15 lH, OH), 3.24-3.49 (mn, 1H, CHCH 2 3.$3-3.72 (mn, 2H, CH 2 OU), 4.36-4.77 (in, 1W, NH). CI MS, in/e 204 -16- N-(tert.-butyloxycarbonyl)-L-isoleucinoI This compound was prepared in 97% yield from the ester 4c (9.84 g? 40 mmol) by reduction with lithium.

borohydride in dry DME analogous to the method described earlier 1 2 TLC Rf 0.47 (eluent MeOH/CHCl 3 7/93). 1

H

NMR CCDCl 3 6 0.83-1.03 (in, 6H, CHCH 3 V CH 2 CH 3 1. 03-1. (in, 2H, CH 2

CH

3

CH

2

CH

3 1.44 Cs, 9H, t-Bu), 2.21-2.61 1H, CHCH 3 3 .32-3. 88 Cm, 4H, CHCH 2 OH) 4. 52-4 .68 IH, NH) CI MS, m/e 218 N- (ter t. -buty loxycarbonyl1) -L-pheny la laninol1 This compound was prepared in 89% yield from tht, ester 4d (11.2 g, 40 inrol) by reduction with lithium borohydride in dry DME analogous to the method described earlier 1 2 TLC Rf 0.50 (e2luent MeOH/QCICl 3 1 7/93).1H NMP, CCDCI 3 6 1 .40 Cs, 9H, t-Bu) 2.CO-2.53 Cm, LH, OH), 2,83 J=5.4 Hz, 2H, CH 2 Ph), 3.33-4.08 3H,

CH

2 OH, CHCH- 2 4.48-4.94 Cbr df IH, NH), 7.22 CI MS, m/e 252 CM++l) N-(tert.-butyloxycarbonyl)- '-prolinol, This compound was prepared in 92% yield from the ester 4g (9.2 g, 40 iniol) h)y reduction with lithium borohydride in dry DME following the procedure that has been described earlier 1 2 TLC Rf 0.81 Celuent MeOH/CHC1 3 7/93). 1 H NMR (coDC 3 5 1.47 Cs, 9H, t-Bu), 1.58-2.18 (in, 4H, CH 2

CH

2 CH), 3.l5-3.66 Cm, 4H, CR 2 N, CH 2 OH), 3.73-4.07 (mn, 3.H, CHN) 4.40-4.80 Cbr s, lH, OH).

CS)-l-hydroxy-O-(p-toluenesulfonyl)-2-amino-N-(tert.-butyloxycarbonyl) -propane C(6a) To a solution of the alcohol 5a (11l.4 mmol) in mL of dry pyridine was added tosyl chloride (12.5 iniol) at -10 0 C. The reaction mixture was stirred at 4 0 C overnight. Af ter completion of the reaction as was 4 -17monitored by TLC (eluent MeOH/CHCl 3 5/95), most of the pyridine was remove,. by evaporation in vacuo at V room temperature. The residue was dissolved in 200 mL of dichloromethane and subsequently washed two times with a 2N KHS0 4 solution to remove pyridine and then with water. The organic layer was dried (Na 2

SO

4 and the solvent was evaporated in vacuo at room temperature.

The crude product was obtained as a white solid in 87% yield. The crystalline product was not purified by recrystallizat,.on because of the instability of this compound.

4 Even at roo.n temperature, in attempts to recrystallize the product, it- decomposes into the cyclic urethane N 0 For 6a; TLC Rf 0.79 (eluent MeOH/CHCl 3 5/95).

Hl NMR (CDC1 3 5 1.15 J=6.5 Hz, 3H1, CHCH 3 1.37 (s,91, 2.44 3H, Ph-CH 3 36-4.11 (in,lH CHCHl 3 3.95 (br s, 2H1, CH 2 OH), 4.53 (br d, 1Hl, NH), 7.22 and 7.73 (ABq, JAB= 8 Hz, 411, Ph-H), CI MS, m/e 300 (M4±4I).

(S)-l-hydroxy-O-(p-toluenesulfonyl)-2-amino-N-Ctert.-butyloxycarbonyl )-3-methyl-butane (6b) This compound was prepared in 70% yield from compound 5b (3,0 g, 14.8 rimol) as described for the synthesis of compound 6a. TLC Rf 0.29 (eluent diisopropyl ether/hexane, 111 NMR (CDCla) 6 0.82-0.98 (in, C11 3

CHCH

3 2.44 3H, pH-CH 3 3.30-3.73 (in, IH, CHCH 2 3.86-4.22 (AB part of ABX spectrami, 2H1, CHCH 2 4.31-4.73 (in, 11, NH), 7.34 and 7.76 (ABq, jAB= 9 Hz, 4H1, Ph-H).

(s)-1-hydroxy-O-(p-toluenesulfonyl)-2-ainino-N-(tert.-butyloxycarbonyl)V3-methyl-pentane (6c) Following the same procedure as used for the preparation of compound 6a, this compound was prepared in 85% yield from compound 5c (5.0 g, 23 minol). TLC Rf 0.27 Celuent diisopropyl ether/hexane, 1H NMR d)5 0.70-1.00 Cm, 6H, CH 3

CHCH

3 Cm 3H, CHCH 2

CH

3 1.40 Cs, 9H, t-Bu), 2.44 3H, Ph-CH 3 3.30-3.80 Cm, 1H, CHCH 2 4.03 and 4.07 (AB part of ABXspctrmd, 2H, CHCH2O), 4.5 (br d, T=4.5 liz, 1H, NH), 7.33 and 7.77 CABq, JAB= 8 1 Hz, 4H1, Ph-H).

(S)-l-hydroxy-o-Cp-toluenesulfonyl)-2-amino-N-Ctert.-butyloxycarbonyl)-3-phenyl-propane C6d) Following the same procedure as used for the preparation of compound 6a, this compound was prepared in 89% yield from compound 5d C5.78 g, 23 rmol). TLC Rf 0.23 CeLi~ent diisopropyl ether/hexane, IH NMR CCDCl 3 6 1.37 Cs, 9H. t-Bu), 2.43 Cs, 3H, Ph-CU 3 2.68-3.00 (AB part of ABX spectrum, m, 2H, CHCH 2 Ph), 3.83-4.22 Cm, 3H, CHCH 2 4.56-4.86 Cm, 1H, NH), 6.98-7.23 C,5,CH 2 Ph-H), 7.32 and 7.4=~s~B 9 Hz, 4H, Ph-H).

Tosylate of N-Ctert. -butyloxycarbonyl)-L-prolincl (6g) This compound was prepared in 99% yield from compound 5g (6.0 g, 30 minol) as described for the synthesis of compound 6a. TLC Rf 0.81 Celuent MeOH/CHCl 3 4/96).

1 H NMR (CDCl 3 (5 13 Cs9Ht-Bu), 1,63-2.12 (Mt, 4H, CH 2

CH

2 CH), 2.44 3H, Ph-CH 3 3.11-3.54 CAB part of ABX spectrum, m, 2H! CHCH2O), 3i76-4.26 (in, 3H1, QH 2

N,

1 30 CH-CH 2 OY, 7,37 and 7.79 CABql JAB= 8 HZ, 411, Ph-H).

C S )-l-thio-S-acetyl-2-amino-N-(Ctert -b~utyIlozycairbon,y2 -propane (7a) To a suspension of cesium carbonate (18.5 g 57 inmol) in 60 mL of dry DMF was added freshly distilled thioacetic acid (9.0 g, 119 inmol). Previously, nitrogen had been passed through the suspenksion for 15 min. When -19- 4 the cesium carbonate was dissolved, the crude tosylate 4 G6a (28.8 g, 96 mrnol) dissolved in 40 mL of dry DMF was added. The reaction mixture was stirred in the dark for 16 hrs at room temperature and was kept under a nitrogen atmosphere. The reaction was monitored by TLC 4 (eluent diisopropyl ether/hexane, After completion of the reaction the solvent was evaporated in vacuo.

The residue was dissolved in, 3U0 mL of dichioromethane and subsequently washed two times with 200 mL 0.5N NaOH solution and onc with water. The organic layer was dried (Na 2

SO

4 and concentrated in vacuo. The crude reaction product was subjected to column chromatography (si3,icagel, eluent. diisopropyl ether/hexane, 1/1) to give 7a in 80% yield. The yield is based on the alcohol 5a. TLC flf 0.37 (eluent diisopropyl ether/hexane, 1/1).

IN NMR (CDC1 3 'S 1,15 (di J=6.5 Hz, 3.H, CH-CH 3 i.44 9Ht 2.35 3H, C(O)CH 3 3,02 (dt J=6 Hz, 2H, CHCH 2 3.940 il-,CC 2 ,4.73 (br d, J=8 Hz, lN, NH). Anal. CalCd. for Cl 0

H

19 11O 3 S: C, 51.48; H, 8.21;, N, 6.00. Found: C, 51.46;i H, 8.30; N, 5.90.

(S)-l-thio-S-acetyl-2-amino-N-(tert.-bui~tyloxycarbonyl)-3methyl-butane (7b) This compound was prepared in 46% yield (based on the alcohol 5b) from compound 6b (3,0 g, 8.25 mmol) as described for the synthesis of compound 7a. TLC R~f 0.40 (eluent diisopropyl ether/hexanie, IN NMR (CDCl 3 'S 0.92 and 0.98 (dd, J=2 Hz, 6, CH 3

CHCH

3 1.45 9H, t-Bu), 1.52-1-98 (in, lH, (!H 3 CHiCH 3 2-36 3H, C(O)CH- 3 2.79-3.14 (AS part of ABX spectrum, lines, 2H, CH 2 3.32-3.78 (in, 11-I CHCH 2 4.25-4.64 (tnt, lH, NH). CI MS, mo/e 262 Anal. Calcd. for C1 2 H2 3

NQ

3 S' C# 55.14; H, 8.87; Nt 5.36. Found: C, 55.41; H, 8.97; N, 5.36.

,4 (S)-l-thio-S-acetyl-2-amino-N-(tert.-butyloxycarbonyl)-3methyl-pentane (7c) Following the same procedure as used for the k preparation of compound 7a, this compound was prepared in 54% yield (based on the alcohol 5c) from compound 6c (7.0 g, 18.9 mmol). TLC Rf 0.46 (eluent diisopropyl ether/hexane, 1 H NMR (CDC13) 6 0.78-1.02 7H,

CH

2

CH

3

CHCH

3 1.02-1.34 2H, CH 2

CH

3 1.43 (s, 9H, t-Bu), 2.35 3H, C(O)CH 3 2.19 and 3.09 (AB part of ABX spectrum, 8 lines, JAX=1.5 Hz, JBX=6.

5 Hz, JAB=14Hz, 2H, CH 2 3.36-3.84 lH, CHCH 2 4.28-4.72 1H, NH). CI MS, m/e 276 Anal. Calcd. for

C

1 3

H

2 5 N0 3 S: C, 56.69; H, 9.15; N, 5.09. Found: C, 56.74; H, 9.17; N, 5.20 (S)-l-thio-S-acetyl-2-amino-N-(tert.-butyloxycarbonyl)-3phenyl-propane (7d) Following the same procedure as used for the preparation of compound 7a, this compound was prepared in 66% yield (based on the alcohol 5d) from compound 6d (8.0 g, 20 mmol). TLC Rf 0,35 (eluent diisopropyl ether/hexane, 1 H NMP.(CDC13) S 1.41 9H, t-Bu), 2.36 3H, C(O)CH 3 2.62-3.21 (2x AB part of ABX spectrum, 12 lines, 4H, CH 2 Ph, Ch 2 3.73-4,20 (m,

CHCH

2 4.37-4.76 1Hi, NH), 7.07-7.40 Ph-H). CT MS, m/e 310 Anal. Calcd. for C 16

H

23 N0 3

S

C, 62.11; 1, 7.49; N, 4.53. Found: C, 61.93; H, 7.51; N, 4.50.

Thioacetate of N-(tert.-butyloxycarbonyl)-L-prolinoI (7g) This compound was prepared in 50% yield (based on the alcohol 5g) from compound 6g (10.3 g, 29 mmol) as described for the synthesis of compound 7a. TLC R 0.56 (eluent diisopropyl ether/hexine, 1 H NMR -21-

(CDCL

3 6 1.47 9H, t-BU, 1.57-2.10 4H, CH 2

CH

2

CH),

2.33 3H, C(O)CH 3 2.84-3.53 4H, CH 2 S, CH 2

N),

3.73-4.09 1H, CHCH 2 CI MS, m/e 260 (ScRs) and (ScS)-l-(chloromethyl)sulfinyl-2-(tert.-butyloxycarbonyl)amino-propane (8a and 9a) These compounds were prepared with some modifications of the procedure described earlier 1 Thioester 7a (14.0 g, 60 nol) and acetic anhydride (6,12 g, 60 mmol) were dissolved in 150 mL of dry dichloromethane. The solution was stirred and cooled to -10 0 C. Subsequently, a solution of 8,9 g of dry gaseous chlorine (the theoretically necessary amount of chlorine was 120 mmol, 8.4 g) in mL of dry dichloromethane cooled at -50 0 C was added via a connecting tube. The temperature of the reaction mixture was kept below 0 0 C. After the addition had been completed, the cooling was removed and the reaction mixture was allowed to warm and was stirred for h at room temperature. The so-prepared sulfinyl chloride precipitated. Subsequently, 200 mL of dry carbon tetrachloride was added and the organic solvents were evaporated in vacuo at room temperature. The residue was stripped with another 200 mL of dry carbon tetrachloride to remove the acetyl chloride.

The thus-isolated sulfinyl chloride was dissolved in 150 mL of dry dichloromethane and added dropwise over a period of 3 hrs to a stirred, cooled and dried (KOH pellets) solution of excess diazomethane in ether.

DUring the reaction the temperature was kept at OOC.

The reaction mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo and the crude product was purified by column chromatography on silicagel (eluent MeOH/CH2C1 2 gradient from 0% to 2% of MeOH) to give the diastereomeric a-chloro sulfoxides 8a and 9a in 41% overall yield. Diastereomer 9a was

I

-22formed in slight excess as was determined from the IH NMR spectrum of the reaction product. Separation of the diastereomeric at-chloro sulfoxides, a tedious task, was achieved by crystallization front ethyl acetate to yield pure 8a, and subsequent column chromatography on silicagel (eluent diisopropyl ether/methanol, 93/7) to yield pure 9a next to a considerable amount of a mixture of Ba and 9a. This procedure was carried out repeatedly.

For Ba: mp 177-178 0 C; TLC Rf 0.23 (eluent diisopropyl ether/MeOH, 93/7). lfi NMR (CDCl 3 6 (d, Hz, 3H, CHCH 3 1.44 9Wf t-Bu), 3,00 ant, 3.17 (AB part of ABX spectrum, 7 lines, JAX=9*0 HZ, Hz, bIB-1 3 .5 Hz, 2H, CHCH 2 3,69-4.35 (in, lH, CHCH 3 4.39 and 4.49 (ABq, JAB= 11 KZI, 2H, S(O)C1 2 C1), 5.08 (br d, lH, NH), CT MS, m/e 256 Anal, Calcd.

for C H 18 NO SCl: C, 42.27; H, 7,09, N4, 54.Fud C 42.30; H, 7.1:3; N, 5.38. CD spectrum (aqetonitrile solution): at 229 nm a single negative Cotton effect was oboerved For 9a,- mp 130 0 C; TLC RE 0.2$ (eluent diisopropyl ether/methanol, 93/7). 1 H NMR (COC1 3 ].1.34 Cd, 7=6. 6 Hz, 3H, CHCH 3 1,42 Cs, 9H, t-Bu), 2.89 and 3.18 part of ABX spectrum, 8 lines, JAXq q. ITZ iBX~ S4 Hz, JAB' 0..5 Hz, 2H, CHCH S(o) 3.79-4. 24 Cm, l14, CH 3 4.57 is, 2H, CS(o)CH 2 CI), 4.73 (hr d, 1W, MH. C! NIS, m/e 256 (M41).Anal. Calcd. for C 9 1I 18

NO

3 3C3, C, 42-27; H, 7,09; N, 5.48. Found: C, 42.04,i H, 7.10;i N, 5.50, CD spectrum Cacetonitrile solution): 4 t 229 nin a single positive Cotton effect was observed(~-0,)

(S

0 Rs) -C (ch'loroMethyl) gulf iuyl-2-(Ctert. -butyloxycarbony amino- 3-methyl-butane (8b) Following the same procedure as used for the preparation. of compounds 8a and 9at this compound waz prepared from Compound 7b (2.3 g, 8,82 mxnol), Due to -23the small difference in Rf-va2'ues of the diastereomeric ~-chloro sulfoxides 8b and 9b unly 120 mg pure 8b could be isolated by combt ned silicagel column chromatography and crystallization from ethyl acetate mp: 180"181*C.

TLC Rf 0.32 (eluent MeOH/CHC1 3 3/97). K NMR (CoDC 3 a 0.99 J=7 Hz, 6H, CH 3

CHCH

3 1.44 Cs, 9H, t-Bu), 1.88-2.14 (in, 1.H, CH 3 CHi- 3 2,84-3.39 CAB part of ABX spectrum, in, 2H, CHCH 2 3.62-4,00 (Mr, IH, CHCH 2 4.44 and 4.48 CABg, Hz, 2H( S(O)CH 2 Cl), 4.76-5.09 (mn, 2.H, NqH). CI MS, m/e 284 (M*1+1J. Anal, Calcd. for

C

11

H

22 NQ3SCl;4 C, 46,$5; H, 7,81; N, 4,94. Found: C, 46.52; Hi, 7. 75; N, 4,76.

15amino-3-tnethy:l-pontane (8c) Thvis compound as prepared in 16% overall yield fromn compound 7c (2.d g, 10 =Qml) as describodi for the synthesis of compounds and 8b. Due to the nlldifference in Rf-values of the diao-toreom~erio A-chloro sulfoxides $o and Oc only 236 mg pure $c could be isolated by column chromatography using silioagel and subsequent crystallization from ethyl acetate, TLC t1f 0.28 Celuent MeOQR-C1 3 3/97). 1H NMR (QC1d 3 0,74-1,05 (mo 11 (sr t-Bu)o 2,8l1-3.24 (AB par~t of ABX Spectrum, m, 2~,CtIFI 2 (Qf..3,77-4405 (in, 114, CHCEC 2 s(Ofl.. 4.44 and dj 1W. NiT).

J

3-phonylpropa1e (86) Following the same procedure as Used for the preparation of compounds 84 and 9a# this compound was prepared In 15% overall yield from compound 76. 3 12.9 nuol). Due to the small difference in Rf-values oc the diastoroomeric, a-chloto sulfoxides 8d and 9d ~e -24only 320 mg pure 8d could be isolated by combined column chromatography using silicagel and subsequent crystallization from ethyl acetate: up 144-146 0 C. TLC Rf 0.30 (eluent MeOH/CHC1 3 3/97). 1 H NMR (CDCl 3 1.42 9H, t-Bu), 1.92-2.85 4H, CH 2 Ph, CHCH 2 4.05-4.37 lHf CHCH 2 ),4.35and 4,49 (ABq, JAB= 11 Hz, 2H, S(O)CH 2 Cl), 5.26-5.36 Cm, lI, NH), 7.25 (br s, SH, Ph-H). CI MS, m/e 332 Anal, Calcd. for Cl 5

H

22 N0O 3 C1: C, 54.29; H, 6.68; N, 4.22. Found: C, 54.39; H, 6.69; N, 4,21.

(SCR

5

)/(SCS

5 )-l(terto-buyloxycarbonyl)-2-(chloromE; nylsulfinylmehyl)pyrrolidine (8g/9g) Following the same procedure as used for the preparation of compounds 8 and 9a, this compound was prepared in 40% overall yield as a mixture of diastereomers from compound 7g (3.3 g, 12,7 mmol). Unfortuunately, attempts to separate the diastereomers failed, TLC Rf (eluent MeQH/CHC1 3 3/97), IH NMR (CDC1 3 Z 1446 Cs, 9H, t-BU), 1.71-2.33 4H. CI 2

CH

2 CH), 2,74 and 2.84 (part of AB part of ABX spectrum, 4 lines, JA k :13 Hz, JAX Or BX9 Hz, CHQH 2 3.023.6 Cm, 6I 2

N,

CHCHa 2 4.02-4,38 Cm, 114 CiCH 2 )f 4.38-4,87 Cm, 2 HS 8(O)CH 2 CI M, exact mass Qa1cd, for C 11

U

4

NO

3 SCet M/e 282.0931 Founds 282.092i, (ScR) and CSS)1,(mthyrthiOmethy ulfiflyl2(trt, butyotxyQarbonyl)aminopropafe (10a and 11a) Following the same procedure as described ealierl, 2 the A-chloro sulfoxideS 8a (3.6 mmol) and 9a (2.0 imol were allowed to react With sodium nethyl mercaptide in ethanol to give the desired compounds l0a and l1a in 98% and 92% yield, reapectively.

For lOa, TLC Pf o018 (eluent ethyl acetate/hexane, 3/1)1H 4MR (CD(13), 94 t(d, 2.33 3tt SC4) 2.8 CHC3),1 1.44 (9 1 OH, t-8U) ,l 2 33 (9,i 3fti SCH3) 2,5 and 3.13

'II

(AB part of ABX spectrum, 8 lines, JX=61 1Z,, Hz, JAB= 1 3.

5 Hz, 2H, CHCH2S(O)), 3.69 and 3.7T5 (ABq, JAB=13.5 HZ, 2H, S(0)CH 2 3.97-4,48 (in OiC 3 4.97-5.40 (in, H, NH). ET MS, m/e 268 Ana2l, Calcd. for C 10

H

2 1 N0 3

S

2 C, 44.92; H, 7.9 2 ,r N, 5.24. Found: C, 45.77; H, 8.04, N, 5.25.

For Ila: TLC Re 0,18 (eltuent ethyl acetate/hexane, 1 H NMR (CDCl 3 6 1,37 J=6.9 Hz, 3H, CHCH 3 1.44 9H, t-Bu), 2.33 3H, SCH 3 2.89 and 3.316 (AB part of ABX spectrum, 8 linest, JAX=7, 2 Hz, J BX=5.4 Hz, JAB13.

5 Hz, 2H, CHCH 2 3.72 and 3.88 (ABq, JAB=l 3 .5 Hz, 2H, S(0)CH 2 3.88-4,33 (in, iF, CHCH 3 5,02 (br d, J=8 Hz, lH, NH). ,V MS, Im/e 268 (MK+l) (ScRs)lI (methylthi omethy1suliny 2tl- -(t.-butYoXycarbonyl)amino-3-methyl'butane, (Ob) This compound was prepared in 88% yield from the a-chloro sulfoxide 8b (120 mg, 0.42 mmo.) by treatment with sodium methyl. mergaptide following the same procedure that has been described earlier 1 2 TrLC Re 0. 21 (eluent MeOH/diisopropy3 ether, 7/93). 'H NMR (C0C13) 0-96 S(d, 7t--6 4 Hz 6H i CH3CHCH3J) .41 (s 9H r tBu) 818-2. 23 n(m, H, CHICHCH 3 2.30 311, Cf 3 2.84 and 3.15 (AB part of ABX spectrum, 8 lines, aAX;:8.3 Hz, JX=3.2 Hz, JAB= 12 9 Hz, 2H, CHC" 2 360-.08 (i HH, CHCE2), 3.68 and 3.74 (ABq, IA#-13, 6 Hz, 2tI S(O)CH 2 5.08 (br j;:3 Hz, lilt NiI). ,I QT S, exact mass calod fr

C

1 2H 26 N03S2, m/e 296.1,354 (M t Found: 296.1358, Anal. Calcd. for 012H 25

NQ

3 S2: C, 48,78; H, 8.53: N, 4,74, Found: C. 48,85; H, 8.90;1 N, 4.75.

(ScP,)--(methylhiomehy)sulfiyln-(el--btylxy darbonyl) amino-3-methyl.-pental (00) Following the same procedure as described earlier 2 the ac-oh3oro sulfoxide 8 (236 mg, 0.7) mol) was allowed i c _i j -26to react with sodium methyl meraptide in ethanol to give the desired compound 10c in 92% yield. TLC Rf 0.65 Celuent MeQH/CC1 3 20/8i.W. IH NMR CCDC1 3 6 0,82-1.09 Cmr 6AII C~!CH 3 C1 2

CH

3 I -1.95 3H, CHCH 3

CH

2

CH

3 1,44 9H, t-Bu), 2.32 3H, SCH 3 2.74-3,38 (AB part of T-RX spectrum, m, 2H, CHCH 2 3.74 21,

S(O)CH

2 C1) 3.83-4.18 1H, CHC- 2 4,94-5.27 1H, NH), Anal, Calcd, for C 1 3

H

27 N0 3

S

2 C, 50.4-; H, 8.79; N, 4.53. Found: C, 49.81; H, 8,83; N, 4.41.

CI MS, exact mass ca1qd. for C 1 3

H

2 8

NQ

3

S;

2 m/e 310.1511 Found: 310.1503.

(SqRs)--Cmethylthiomethyllsultinyl-2-(tert,-butyloxycarbonyl)amino-3-'phenyl-p'opane Following tha same procedure as described earlierl, 2 the a-chloro sulfoxide 8d (310 ng, 0.94 mmol) was allowed to react with sodium methyl mercaptide in ethannL to give the desired compound ld in 76% yield. TLC Rf 0,26 (eluent ethyl acetate/hexane, 1 H NMR (CC13) S L.42 9I, t-5u), 2.31 3H, SCH 3 2,74-3.26 4H, CHCH 2 S(OI, CH 2 Ph), 3.71 2H, S(Q)CH 2 4,04-4.43 1H, CHCH 2 5.17-5.44 1IHI NH), 7.24 Cs, SH, Ph-H). CI M$J, m/e 344 Anal. Calcd, for C 1 0 2 00NO 3

S

2 1/2H 2 0: C, 54,52 Hl 7,15; M, 3.97. Found: C, 54.61; H, 7.33; N, 3.82, CSc 5 )-l-(nbutythionethyl)sulfinyl-2-(teLrt.-butylxycarbonyl)aamino-propane This compound was prepared in 95% yield from the c-chloro sulfoxide 8a (1.17 g, 4.6 imol) by treatment with sodium butyl mercaptide which was prepared in situ by reaction of sodium (4,6 mmol) with freshly distilled 1-butane-thiol (5.0 imol) in 20 mL of ethanol. The reaction mixture was worked up following the procedure that has been described earlierl 1 2 TLC Rf 0.42 (eluent MeOH/CHCL 3 i 3/97). 1 H NMR (CDC'1 3 6 0.93 J=6 Hz, 3H CH 2

CH{

3 -27- 1 1.23-1 .80 Cm, 4H, CH 2

CH

2

CH

3 1 .41 CD, J=6 Hz 3H, CHCH 3 1. 44 9H, t-Bu) 2 .74 Ct, J=7 HZ, 2H, SCH 2 CH-, 2.82 and 3.20 (AB part of ABX spectrum, 8 lines, JAX= 7 0 Hz, JBX= 4 4 Hz, JAB= 1 3 0 Hz, 2H, CHCH 2 3.76 (s, 2H, S(O)CH 2 4.02-4.43 Cm, 1H, CHCH 2 9.06-5.41 Cm, IH, NH). CI MS, exact mass calcd. for Cl 3 t- 2 8

N

3

S

2 M/e 3L0.1511 Found: 310.1512. Anal, Calcd. for C 1 3

H

2 7 440 3 S2: C, 50.45; H, 8.79, N, 4.53. Found: C, 50.22; Hei 8.76, N, 4.44.

CScPS)-l-fl-octylthiomethyl)sulfinyl-2-tert.-butyloxycarbonyl )amino-propane This compound was prepared in quantitative yield from the cI-chloro sulfoxide Sa C315 mg, 1.23 nunol) by treatment with sodium octyl mercaptide which was prepared in situ by reaction of sodium (1.35 mmol) with freshly distilled 1-octanethiol (1.48 mrnol) in 10. mL of ethanol.

The reaction mix'ture was worked, up following the procedurc that has been described earlier 1 2 TLC Rf 0.57 Celuent MeOH/CHCl 3 20/80). IH NMR CCDC1 3 5 0.81 (br J=6 Kz, 3H4, CH 2

CH

3 1.20 Cb(,1W CH 2 5

CH

3 t~1) 1,29 Cs, part of do CKHC- 3 1.37 Cs, 914, t-Bu), 1,40-1.78 Cm, 21{, SCH 3

CH

2 )j 2.66 Cto J=7.2 Hz, 2H, SCH 2

CI{

2 2,71 and 3.09 CAB part of ABX spectrum, 8 lines, JAX= 7 2 Hz, JBy=15.4 Hz, JAB=l.2.9 Hz, 02Wo CHCH 2 SSQ)), 3.68 (s, 2H So)H 2 ),3.91-4.31 (in, fW WW) 5.31 Cbr do J=7 Wz, NH). Anal. Calcd. for Ci 7

H

35

NQ

3

S

2 sI./2H 2 0: C, 54.51 H, 9,42; N, 3.74. Found: C, 54.23; H, 9,28; N, 3.36.

CScRs) and CScSs)-1-(tert.-butyloxycarbony1.)-2-(methylthiomethylsUlfiflylmethY1)pyrrolidile (l~g and hlg) These compounds were obtained in 88% yield as a mixture of diastereomers from the a~-chloro sul3f oxide 8g (590 mg, 2.1, mmol by treatment with sodium methyl -28mercaptide in ethanol following the procedure that has been described earlier 1 2 By careful column chromatography using silicagel two fractions weighing 278 mg (fraction 1: 45%) and 264 mg (fraction 2: enriched in compound lOg and llg, respectively, were obtained. The ratio of compounds log and hg in beth fractions is unknown.

The NMR spectra of both fractions were almost identical.

TLC Ref 0.51 (fraction 1) and 0,47 (fraction 2) (eluent MeOH/CHCl 3 3/97). LU NMR (CDC1 3 6 1.41 9H, t-53u), 1.66-2.17 Cm, 4H, CH 2

CH

2 CH), 2.28 3H, SCH 3 2,49-3-47 (in, 4H-, CHCH 2

CK-

2 3.61 and 3.72 CABq, JjkB=1 3 .6 Hz, 2H, S(O)CH 2 3.94-4.36 (in, 1H-, CHCH 2 CI MS, m/e 294 (ScRs) and (SCS 5 )-l-(methylthiomethyl)sulffinyl-2-amino-propane (12a and 13a) Following the same procedure as described earlier 1 2 the Boc amino protecting group of compounds 10a (2.6 mmol) and Ila (0.63 mniol) was removed by treatment With trifluoroacetic acid at 000-- Stubsequent ion-exchange column chromatogrzaphy on Amberlite IRA-410 yielded compounds 12a and 13a in 93% and 95% yield, respectively.

For 12a: TLC Re 0.22 (eluent MeOH/CHCl3, 10/8Q).

1NMR (CD 2 Cl 2 6 1.28 J%-6.3 Hz, 3H, CHCH 3 2.31 3H, SCH 3 2.81 and 2.87 (AB part of ASX spectrum, lines, JAX= 8.0 Hz, JBX= 4 9 Hz, 1.,AB= 12 8 Hz, 2H-,

CHCH

2 3.11 (br s, 2H, NK 2 3.33-3.73 Cn. IlH,

QHCH

3 3.73 and 3.80 CA~q, JAB'13.5 Hz, 2H, S(o)CH 2

S)-

For 13a: TLC Re 0.22 Celuent tMeOH/CH-C1 3 2 1 H NMR (CD 2 C12) r$ 1.28 7- 6.3 Hz, 3H, CHCH- 3 1.97 2H, NH 2 2.33 Cs, 3ti, SCH- 3 2.84 and 2.93 (ABl part of ABX spectrum, d, 2H, CHCH",S(0)), 3.46-3.71 Cm, 1.H, CHCH- 3 3.72 and 3.84 (ABq, J ,3l3.5 liz, 211, S(Q)CH 2

S).

(ScRs)1-(methylthionethyl) sulfilyl-2. 3-methyl -butane -29- This compound was prepared in quantitative yield from compound l0b (110 mg, 0.37 mmol) by treatment with trifluoroacetic acid at 0 0 C and subsequent ion-exchange column chromatography on AmberJlite IRA-410 following the procedure that has been described earlier 1 2

TLC

Rf 0.59 (eluent MeOH/CHCl 3 20/80). 1 H NMP. (CDCl 3 6 0.84 and 0.92 (dd, J=2.3 Hz, 6H, CH 3

CHCH

3 1.43 (br s, 2H, NH 2 1.43-1.84 (in, 1H, CH 3

CHCH

3 2.26 (s, 3H, SCH 3 2.68 and 2.74 (AB part of ABX spectrum, doublet, J=1.5 Hz and singlet, respectively, 2H, CHCH 2 2.93-3.28 1H, C1ICH 2 3.59 and 3.77 (ABq, JAB:; 13 Hz, 2H, S(O)CH 2

S).

(Sc 0 ls)-l-(methylthiomethyl)sulfinyl-2-amino-3-methyl-pentane (12c) Following the same procedure as described earlier 1 2 the Boc amino protecting group of compound 10c (207 mng, 0.67 minol) was removed by treatment with trifluoroacetic acid at 0 0 C and subsequent ion-exchange column chromatography on Ainberlite IRA-410 to yield compound 12c quantitatively. 1 H NMR (CDCl 3 6 0.81-1.10 (in, 6H, CHCH 3

CH-

2

CH

3 it, 1.10-1.72 (in, 3H, CHCH 3

CH

2

CH

3 1.48 (br s, 2H, NH2), 2.33 3H, SCH 3 2.73 and 2.82 CAB part of ADX spectrum, 2H, CHCR- 2 3,11-3.43 1W, CHCH 2 3.68 a,,d 3.86 (ABq, JAB= 13 5 Hz, 2H, S(O)CH 2

(S

0 I (methylthiomethyl )sulf iny1-2-amino-3-pheny.-propane (12d) This compoundl was prepared in 73% y-1d from compound 10d (244 mng, 0.71 mm'ol) by trettmant with trifluo acetic acid at 0 0 C and subsequent ion-exchange column chromatography on Amberlite IRA-410 following the procedure that has been described earlier 1 2 1 H NMR (CDC1 3 6 1.38-1.82 (br s, 28-, NH 2 2.31 3H, SCH 3 2.58-3.19 (mn, 4H, CH 2 Ph, CHCH 2 3.50-3.87 (in, l1H, oCHH 2 3.70 2H, S(O)CH 2 7.04-7.47 (in, 5H, Ph-H).

CScRs)lI(lbutylthiomethyl)sulfinyl-2-amino-propane (12e) Following the same procedure as described earlierl' 2 the Boc amino protecting group of compound 10e (1.33 ij g, 4.3 minol) was removed by treatment with trifluoroacetic acid at 0 0 C. Subsequent ion-exchange column chromatography on Amberlite IRA-410 yielded compound 12e quantitative& y.

TLC Rf 0.61 (eluent MeOH/CHCl 3 20/80). 1 H NMR (CDCl 3 6 0 .92 J=5 .4 Hz, 3H, CH 2

C-H

3 1 .28 J=6 .4 Hz 3H, CHCH 3 1.34-1.87 (in, 4H, (CH 2 3

CH

3 2.58-3.08 4H, CHCH 2

NH

2 3.31-3.91 Cm, 1H, CHCH 3 3.77 2H, S(O)CH 2

S).

(ScR 5 )-l-(n-octylthiomfethyl)sulfinyl-2-amino-propane (12f) This compound was prepared in quantitative yield from compound 10f (434 mg, 1.23 rnmol) by treatment with trifluoroacetic acid at 0 0 C and subsequent ion-exchange column chromatography on Amberlite IRA-410 following the procedure that has been described earlier 1 2

TLC

RF 0.57 Celuent MeOH/CHCl 3 20/80). 1H NMP. CCD 2 Cl 2 60.88 Cbr t, J=5 Hz, 3H, CH 2

CH

3 )t 1,28 (br s, 13H,

(CH

2 5

CH

3

CHCH

3 )t 1.33 part of d, CHCH 3 1.48-1.84 I (me 2H, SCH 2

CH

2 2.04 2H, NH 2 2.56-3.31 Cm, 4H{, CVCK2S(O) SCH 2

CH

2 3.42-3.84 IH, CHCH 3 3.80 s 211,t S(0) CH 2 S) CSpRSO and CScSs)-2-(methylthiomethylsulfinYlmethyl)pyrrolidine (12g and 13g) These cbmpounds were prepared in 97% and 92% yield from compounds lOg (fraction 1, 278 mg, 0.95 minol) and llg (fraction 2, 264 vmg, 0.90 mrnol), respectively, by treatment with trifluoroacetic acid at 0 0 C and subviequent ion-exohav.4e column chromatography on Amberlite, IRA-410 following the procedure that has been described earlierl' 2 For fraction 1: IH NM?. (CDCl 3 5 1.40-2.42 Cm, 4H, CH 2

CH-

2 CH), 2.23 Cs, 3H, SCH 3 2.61-3.48 Cm, 4H,

CHCH

2

CH

2 3.48-3.92 (mn, 1H, CHCEI 2 3.65 and 3.93 CABq, JAB= 13 5 Hz, 6fI ~QC 2 .27 (br 1H, 14H) CI MS, m/e 194 CM++l) For fraction 2: 1 H NMR CCDCl 3 6 1.43-2.08 Cm, 4H, CH 2

CH

2 CH), 2.21 Cs, 1H, NH), 2.33 Cs, 3H, SCH 3 2.76-3.17 Cm, 4H, CHCH 2 SCO), CH 2 3.43-3.83 Cm, 1H,

GHCH

2 3.67 and 3.95 CABq, JAB= 13 5 Hz, 2H, SCO)CH 2

S),

CScPs)"1-(iethylthioiethyl)sulfiny-2- L8 uracilyl)acrylanidoTl-propane C(2a) This compound was prepared by a mixed anhydride coupling procedure. To a solution of 6-methyl-uracil acrylic acid 14 C784 mrg, 4.0 mmol) in 5 mL of DMF at 0 0 C, was added triethyl amine C4Q4 mng, 4.0 mmol) and isobuty. chloroformate (544 mg, 4,0 mmol), After 5 min, a solution of 12a C664 mg, 4.0 mmol) in 10 mL of QMF was added. The reaction mixture was stirred in the dark at 0 0 C for 16 hrs. Evaporation of the solvent and gel filtration on Fractogel TSK tiW-4OCF) Celuent MeQH/H 2 0, 85/15) afforded the desired compound 2a in 71% yield (based on the Ot-chloro sulfoxide Wa. TLC Rf 0.53 Celuent MeOH/CHCl 3 20/a0). 1 H MR (D 2 0) 1,37 Cd, J=6.0 Rz, 3HCHC 3 ),2.26 3H, SC 3 2.37 3H, CC6)-CH3), 3.11 and 3.19 (AB part of ABX spectrum, 5 lines, JAX= 5 4 Hz, JBX= 3 6 Hz, 'JAB 1 3 5 Hz, 2H, CHCH 2 3.90 and J4.06 48ABll- 5 tRZ, 2H, $CQ)Ct1 2 S)t 4.31-4.62 (mi, 114, CHCH3), 7.00 ar~d 7,30 CA~q, JAB=16, 0 Hz, 2H5, CH=CH), FAB MS,. m/e 34 N2Dt +770a Cc=0.130, MeOli/H 2 0, Anal. Calcd, for C 1 3 11 19

N

3 0 4

S

2 C, 45.20; Hf 5.54; N, 12.16. Found: C, 45.24; H, 5.531- N, 11.84.

(ScSs) (,-(mthylthiomethyl)8ulfinlY2- -32uracilyl)acrylamidol-propane (3a) Following the same procedure as used for the preparation of compound 2a, this compound was prepared in 39% yield (81 mg) from compound 13a. The reaction product obtained was approximately a 75 25 mixture of compounds 3a and 2a. This was determined by integration of the AB quartet signals for S(O)CH 2 S for compounds 2a and 3a in the 1 H NMR spectrum of the reaction product.

TLC Rf 0.53 (eluent MeOH/CHC1 3 20/80). 1 H NM, (D 2 0) 6 1.37 J=6.6 Hz, 3H, CHCH 3 2.28 3H, SCH3), 2.40 3H, C(6)-CH 3 3.16 and 3.23 (AB part of ABX spectrum, 8 lines, JAX=8-1 Hz, JBX= 4 5 Hz, JAB= 1 3 Hz, 2H, CHCH 2 3.96 and 4,12 (ABq, JAB= 1 3 5 Hz, 2H, S(O)CH 2 4.31-4.57 1H, CHCH3), 7,00 and 7,30 (ABq, JAB= 16 .0 Hz, 2H, CH=CH). FAB MS, m/e 346 [(41250= +630 (c=0.10, H 2 Anal. Calcd. for C 13

H

19

N

3 0 4

S

2 2H 2 0: C, 40.93; H, 5.02; N, 11.02. Found: C, 40.94; H, 4.78; N, 11.18.

(S

0

R

5 )1-(methylthiomethyl)sulfinyl-2-[$-(6-methyl-5uracilyl)acrylamido] -3-methyl-butane (2b) Following the same procedure as used for the preparation of compound 2a, this compound was prepared in 29% yield (40 mg) from compound 12b (0.37 mmol).

The reaction product obtained was approximately a 75 mixture of compounds 2b and 3b. This was determined by integration of the AB quartet signals for CH=CH as well as from the singlet signals for SCH 3 for compounds 2b and 3b in the 1I NMR spectrum of the reaction product.

TLC Rf 0.50 (eluent MeOH/CHC13, 20/80), 1l NMR (CD 3

OD)

6 1.04 J-'6.6 Hz, 6H, CH 3

CHCH

3 )1 1.80-2.18 11,

CH

3

CHCH

3 2.34 (for 3b: 2.37) 3H, SCH 3 2.41 (s, 3H, C(6)-CH 3 2.39-3.22 (mi, 2, CHCH 2 3.89 and 4.04 (ABq, JAD= 13 8 HZ, 211, S(O)CH 2 4.23-4.52 (m, 1H, CHCH 2 7.29 and 7.49 (for 3b: 7.28 and 7.48) (ABq, JAB=15.3 Hz, 2H, CH=CH). FAB MS, m/e 374 +920 (c=0.20, MeOH/H20, (ScRs)-l-(methylthioethyl)sulfinyl-2- uracilyl) acrylamido4-3-methyl-pentane (2c) Following the same procedure as used for the preparation of compound 2a, this compound was prepared in 20% yie.1d (50 mg) from compound 12c (0.67 mmol). TLC RfO.52 (eluent MeOH/CHC13, 20/80). 1H NMR CCD 3

OD)

6 0.79-1.09 6H, CHCH 3

CH

2

CH

3 1,09-1.91 3H.

CHCH3, CH 2

CH

3 2.32 Cs, 3H, SCH 3 2.39 Cs, 3H, C(6)-CH 3 2.76-3.24 2H, CHCH 2 3.85 and 3.99 (ABq, JAB= 14 Hz, 2H, S(0)CH 2 4.28-4.61 lH, CHCH 2 7.26 and 7.45 (ABq, JAB= 1 5 5 Hz, 2H, CH=CH)I FAB MS, m/e 388 25D= +920 (c=0.064, MeOH/H 2 0, Anal.

Calcd. for C 1 6

H

25

N

3 0 4

S

2 .l 1/2H 2 0: C, 46,36; H, 6.08; N, 10.14. Found: C, 46,29; H, 5.94; N, 9.71 (ScRs)-l-(methylthiQmethyl)sulfinyl)-2- uracilyl)acrylamidol-3-phenyl-propane C2d) Following the same procedure as described fcr the preparation of compound 2a, this compound was prepared in 51% yield (112 mg) from compound 12d (126 mg, 0.52 mmol), TLC RE 0.81 Celuent MeOH/CHC1 3 20/80). IH NMR (C0 3 QD) 2.30 3H, SCH 3 2.37 3H, C(6)-CH3), 2428-3.26 Cm, 4H, CHCF{ 2

CH

2 Ph), 3.83 and 3.97 (A~q, JPa 1 3 7 Hz, 2H, (0)CH 2 4.49-4.82 (i tH, CHCI), 7.19 and 7.41 ABq, JAB= 1 5 3 Hz, 2H, CH=CH), 7.29 (so 511, Ph-H). FAB MS, n/e 422 131 25D= +210 (c=0.06, MeOH/H 2 0, 1/1)i Anal. Calcd6 for C1 9

H

23

N

3 0 4

S

2 'l/2H 2 0: C, 51,92; H, 5.27;' N, 9.56. Found: C, 52,75; H, 5r.50; N, 9,204 (Scs).ll(nibutylthiomethyl)sulfinyl-2- uracilyl) acrylamido3-propane (2e) This compound was prepared in 37% yield (620 -34mg) from compound 12e (1.33 g, 4.30 mmol), following the same procedure as used for the preparation of compound 2a. TLC Rf 0.66 (eluent MeOH/ClIC1 3 20/80). 1 H NMR (CD 3

OD)

6 0.77 (br t, J=6.3 Hz, 3H, CH 2

CH

3 1.20 J=6.9 Hz, 3H, CHCH 3 1.10-1.68 4H, CH 2

CH

2

CH

3 2.18 (s, 3H, C(6)-CH 3 2.60 J=;7.2 Hz, 2H, SCH 2

CH

2 2.79 A and 3,03 (A8 part of ABX spectrum, JAX=9.3 Hz, JBX=4.4 Hz, JAB= 1 3 0 Hz, 2H, CHCH 2 S(Ofl, 3.70 and 3.83 (ABq,

JAB=

13 6 Hz, 2H, S(O)CH 2 4,21-4.57 LH, CHCH 3 )e 7.02 and 7.27 (ABq, JAB= 1 5 .0 Hz, 2H, CH=CH). FAB MS, m/e 388 K1 2 ,50 +800 0.ll5, Meat/H20, 1/1).

Anal, Calcd. for C 1 6

H

2 5

N

3 0 4

S

2

.H

2 0: C, 47.39; H, 6.21; N, 10.36. Found: C, 47.47; H, 6.47; N, 10.32.

(ScRs) l-(f-octylthiomethyl)sulfinyl-2 E (6 uracilyl)acrylamido]-propane (2f) Following the same procedure as described for the preparation of compound 2a, this compound was prepared in 50% yield (272 mg) (based on the 'chloro sulfoxide 8a) from compound 12f (340 mg( 1.24 mmol). TLC Rf 0.71 (eluent MeOH/CHCl 3 20/80).

1 H NMR (CD300) *i 0,91 (br z, J=5.5 Hz, 3H, CH 2

CH

3 1.28 (br s, l0, (CH 2 5 Cf3), 1,37 J=8.0 Hz, 3H, CICH 3 1.52-1.82 2H, SCH 2

CH

2 2.35 3H, C(6)-CH 3 2.77 J=7.0 Hz, 2H, SCH 2

CH

2 3.30 J=5.0 Hz) and 3.17 J=9-0 Hz, AB part of ABX spectrum, 2H, CHCH 2 3.$8 and 3.95 (Aq, JAB= 13 Hz, 2H, S(Q)CH 2 4.42-4.51 11, CHCH 3 7.18 and 7o42 (Afqe 3

AB=

1 6 0 Hz, 2H, CH=CH4). FAB MS, info 444 [CCJ 25D= +930 (c-0.102, MeOH), Anal. Calcd. for

C

0

H

33

N

3 04S2: C, 54.15; Ht 7.50; N, 9.47. Found: C, 53.92; H, 7.53; N, 9.21.

(ScRs) and (ScSs)-l- [8.(6-methyl-5-uraoilyl)acrylamide}" 27-(methylthiomethylsulfinylmethyl)pyrrolidine (2g and 3g)

-A

These compounds were prepared in 36% yield (123 mg) and 72% yield (209 mg) from compounds 12g (fraction 1, 178 mg, 0.92 mmol) and 13g (fraction 2, 150 m'g, 0.78 mmol), respectively, following the same procedure as used for the preparation of compound 2a.

For 2g: TLC Re 0.58 (eluent Me0H/CHCl 3 20/80).

'H NMR (CD 3 OD)al .98-2, 2 9 (in, 4H1, CHCH 2 CH), 2.38 3H, SCH 3 2.41 3H, 2.98-3.56 (mn, 2H, CHCH,S(0)), 3,56-3.90 (in, 2H, CHN), 4.06 and 4.34 (ABq, JAS=1 3 8 Hz, 2H, S(0)CH 2 4.47.4.77 (mn, 18, OCHH), 7.53 2H, CH#CH). FAB IIS, ni/e 372 (XM+1).

uracilyl)acrylainidojpr~pare (2h) This compound was prepared directly from 8a without purification and characterisation of the Intermediates and 12h, The procedures employed were identical to those described for the Synthesis of 2a; however, sodium ethyl mercaptide was sed in the conversion 8a 10h, The overall yield was 60% when the reactions were carried out on a scale of 2.35, imol, starting with 8a, TLC Re 0,55 (eltient Me0H/CHCl,, L11 NIIR (D,,0)6 1.19 J=.7.5 Hz, 3H, 1,33 J=7.2 Hz, 3H, C8CH 1 2,33 3H1, CH0H,), 2.65 and 2i79 1 =7,3 Hz, 2H, CH2CH,)t 1.96 and 2.10 (AB part of ABX spectrum, 8 lines, J4 10 1 1z, JL,,3.4 Htz, JAB",5. Hz, 21H, CIICHUS(0)), 3.94 and 4.08 (ABq, J45t:14.1 Hz, 2H, S(Q)CHjS), 4,33-4o66 (in, 18, 08082), 6.96 and 7.53 (ABq, JA6= 1 5 o 5 Hz, 2Hf, CII CH). FAB MS$ m/e 360 K4+1I)f 284 CttUtN'0,,S), 268 C ItHIA10',QS), 236 C 1 ~f8I 4

NIQ

3 181 6 8~N0S) ,179 C8HN 03)t 165 j.,S) 153 CiN0)75 (227%t C H 7 tc.I2 D+69Q (a 0,145, The biological activity of the present compounds has been investigated by jIn vitro clonogenic assay, The in vitro clontogenic assay was effected in leukemia 1. 1210 cells in soft agar medium There -36is a good correlation between the activity in vitro and that in vivo of the medicaments investigated. On the basis of the findings in the in vitro analysis, therefore, the in vivo activity can be predicted with good results. In the analysis, the inhibition of the L 1210 colony formation by sparsomycin and derivatives thereof is determined for various concentrations, and the dose giving 50% inhibition of the colony formation, as compared with non-treated control cells is calculated

(ID

50 The analysis is carried out as follows (variant of the method described in Cancer Chemother. Rep, (1961), 51, 451).

From a suspension culture one hundred L 1210 cells are plated out in 35 mm culture dishes (Falcon) containing i ml soft agar culture medium and the compound to be investigated in suitable concentrations. The soft agar culture medium consists of Dulbecco's medium supplemented with 20% horse serum, 60 /umole 2-mercaptoethanol, mg/ml L-asparaqine, 75 mg/ml DEAE dextran (molecular weight 2 x 106) and 0,3% bacto agar (Difco), The culture dishes were incubated at 370C in an atmosphere of

CO

2 in humiditied air for 3 days. After this period of continuous exposure to the medicament the colonies were counted and dose-effect curves made. From these curves, the medicament dose is calculated Which causes inhibition of the colony formation as compared with non-treated control cells.

The results are summarized in the following A Table i. It appears therefrom that the inhibition of L 1210 colony formati'. by compounds (2a) and (2f) is ol the same level as obtained with n-octylsparsomycin and benzyl-sparsomycin, The low activity for compound (3a) proves that the chirality at the atom of the sultoxide group is important for the antitumor activity.

r~ -1~~uli- -37- TABLE 1 L1210 clonogenic assay (1pM) Sparsomycin 0.47 n-pentyl-sparsomycin 0.06 benzyl-sparsomycin 0.11 n-octyl-sparsomycin 0,10 (2a) ala-sparsomycin 010Q (2h) ethyl-ala-sparsomycin 0,15 (2e) n-butyl-alasparsoiyoin 0,05 (2f) n-octyl-ala-sparsomycin 0.03 (3a) S-epimer-ala-sparsonycin 0155 The activity of the nvel ompounds was also measured in two cell-tree systems, namely saccharomyces cerevisiae and E. coli, a prokaryotic and a yotkarytia system, respectively, The peptidyl transferase activity was measured by the potyphenylalanine synthesis assay (Nireanber and Natthei, Proc.NatlAoadSci. USA 47 (1961) 1588), the fragment reaction (Monr~o Methods Enzyrnol- 20 (1971) 472) and the puromycin reaction, in all the in vitro tests, i.e. PolyPho synthesis, £raqmrxnt reaction andl puromycin reaction, ribosomes from 8. ci MVRE.600 and S, cereviiae Y166 were Used. The reaction mi~xturo for Polyphenyalanin synthesis Was Iightlydifterent for E. coll. and S. cerevisiao, E, coli,, The reaction mixturte (50 /ul) contained $Q mM Tris-ICi pR 7.61 15 mM MC 2 ,0 0 1t KC1., 15 mM a-mercapto ethanol, mM GTPo 10 mM ATt' 0.Z Mg/ml of polyuridylic acid, 1.5 mg/ml tRNA, 0.3 uR of ribosomos mc/ml of phosphoenol pyruvateo 20 /ug/ml of pyruvate kinase, the required concentration of sparsomycin or its analogue and 5 1 Of supernatant fraction S-100, Subsequently, [3tHIphenylalanine# 30 /uM (about 60 cpm/pmol) was added to start the reaction. incubation was at 370C for 30 min, The reaction was stopped by the additiorn -38of 1 ml of 10% TCA and the samples were filtered through glass fiber filters. The filters were washed with ml of cold 10% TCA, dried and counted for radioactivity.

S. cerevisiae; The test Was carried out as indicated above for E, coli, The phosphoenol pyruvate and pyruvate kinase were, however, replaced by creatine phosphate mM) and creatine phosphokinase (50 /ug/ml). The incubation was performed at 30 0 C for 30 min and the samples were processed as described above, Under these conditions the control samples polymerized 5-14 and 3-8 molecules of phenylalanine per ribosome derived from E, coli and So cerevisiae, respectivelyo Ribosomes and supernatant factors from coli and So cerevisiae were prepared according to standard procedures, described by Staehelin and Maglott Methods Enzymol. 20 (1971) 449, and by Sanchez-Madrid et all Eur,!,Biochem 98 (1979) 409.

The fragment reaction was carried out in 1 0 /ul of 33 mM Tris-HC1 pH 7,4t 270 mM KCI, 13 mM magnesium acetate containing I mq/ml of ribosomes, 2mM puromycin, 1 pmol of N-acetyl-L3FtbLU-ACCAC(U), the required concn tration of sparsomycin or its analogue and 33 methanol.

The reaction was initiated by tho addition of the alcohol and allowed to proceed at QC for 30 min, and was then stopped by the addition of 100 /ul p E 0.3 M sodium acetate PH saturated with M4SO 4 The samples were extracted with 1.5 ml of ethyl acetate and 1 ml of the organic phase was checked for radioactivity.t The 3' terminal Cntaiaucleotido N'-acetyl- [31teuU-tCAQ(U) was prepared from N-aoetyt- [3R1beulttRwA by rlbonuclease TL is described by Monro, Tho puronycin reaction was carried out in 2$ /ul of 30 mM XC1I $0 mA NH4Cl 30 mM Trist10l pH 7.8, mM, MqCl2, 10 pmol of tiboome, 100 /ug/nl of poly(U) and 15 pmol of N-acetyl- [3 Phe-tRNA. The reaction mixture 39 was incubated for 30 min at 370C and then 1 /ul of 20 mM puromycin was added and incubation was followed for 5 more min. To the samples 250 /ul of 0.1 M sodium carbonate and 0.7 ml of ethyl acetate were added, shaken for 1 min, centrifuged to separate the phases and 0.5 ml of the upper organic phase taken for estimating the radioactivity.

The results are summarized in the following table 2.

The concentration of sparsomycin or an analog giving inhibition of the protein synthesis relative to the control is given as ED 5 L i i r TABLE 2 in vitro peptide bond formation assays: EDrO polyPhe syntres- s E.coli S. cerevisiae Fragment reaction E.coli S. cerevisiae Puromycin reaction Ecoli Sparsoiuycin 8.5 5.2 3.2 2.8 0.1 n-octyl-sparsoinycin 1.6 1.2 60 300 0.25 benzyl-sparsomycin 0-6 0.6 7.6 2.6 0.13 (2a) Ala-sr'-rsomyc;,a 14.6 50 18 39 0.25 (2h) Ethyl-Ala-sparsouiycin 12 11-1 10.3 10.4 0.07 (3a) S-epimer Ala-sparsomycin 283 143 55 68 2.7 a mixture of diastereomers from the a-chloro sulfoxide 8g (590 mg, 2.1 mmol) by treatment with sodium methyl i i 39b It will be apparent from the results given in table 2 that ala-sparsomycin (2a) and in particular ethyl-alasparsomycin (2h) are effective inhibitors of protein biosynthesis, although less effective than sparsomycin itself and the n-octyl and benzyl derivatives.

Inhibition of bacterial growth was tested in solid as well as in liquid medium. For the test on agar plates, petri dishes containing 15 ml of LB medium g/l bactotryptone, 5 g/1 yeast extract and 5 g/l NaC1) i were covered with 5 ml of the same medium kept melted at 40 0 C containing 40 /ul of a culture of the required j 5 bacterium at approximately an A 5 5 0 of 2. After solidification, S3 mm filter disks containing 4 /ul of a 10 mM solution j of the antibiotic in 50% EtOH/DMSO were placed on the surface of the agar and the plates were incubated at i 37 0 C for 24 h. For the test in liquid medium, E. coli j 10 MRE600 was grown in LB medium supplemented with 0.2% of glucose to exponential phase (A 560 0.1) and distributed in 1 ml aliquots in tubes containing the required amount of antibiotic. The tubes were incubated at 37 0 C for 4 h, subsequently diluted with 3 ml of 0.1% of sodium azide to stop growth and the absorption at 550 nm was measured. The absorption of the medium without addition of antibiotic was considered as 100% growth.

The results are summarized in the following table 3.

TABLE 3 Growth inhibition E.coli MRE600 Sparsomycin 11 n-octyl-sparsomycin benzyl-sparsom .cin 1 (2a) Ala-sparsomycin 5.6 (2h) Ethyl-ala-sparsomycin (3a) S-epimer Ala-sparsomycin 44 Fron table 3 it is apparent that ala-sparsomycin and ethyl-ala-sparsomycin are also effective inhibitors of bacterial growth, superior to sparsomycin itself but less active than the n-octyl and benzyl derivatives.

The results obtained in the assays to '-termine 1 1 1 .j -41the inhibition of protein synthesis demonstrate that the assay systems concerned have little or none predictive value for the antitumor activity (L 1210 assay system) of compounds.

Toxicity The LD50 values lethal dose for 50% of the animals) are given in the following table 4. The experiments were performed on BCBA/Rij mice, 10 per dose levels, 4-5 dose levels. Treatment was administered daily ip. during nine consecutive days.

TABLE 4 values for sparsomycin analogues (95% confidence interval) Drug: LD50 mg/kg/inj (95% confidence interval) sparsomycin 0.26 0.17 0.38) n-pentyl-sparsomycin 4.7 3.7 5.9 benzyl-sparsomycin 5.0 4.0 6.3 n-octyl-sparsomycin 29.5 (24.0 37.0 (2a) ala-sparsomycin 2.33 1.8 3.2 (2h) ethyl-ala-sparsomycin 8.0 6.95 9.2 Antitumor activity: The results are shown in tables 5, 6, 7 and 8 and are expressed as a treatment to control ratio of the median respective survival times. In the tables, information is provided about the model used, treatment and number of survivors after day 60 of the experiment. All experiments were performed on male mice.

V -42- TABLE Antittumor activity of Sparsomycin analogues on L1210 leukemia 4 (0p on day 0 in CD2F1 mice. Treatment ip on days 1-9.

Drug: optimal dosea: Median T/C R~ange: LTS/totalb ~mg/kg/iM Sparsomycin 0.25 125 0/18 n-pentyl-sparsrnycin 5,4 164 154-175 2/42 benzyJ.--sparsomycin 6.4 141 110-'171 0/96 n-octyl-sparsomycin 15.0 136 120-152 1/78 (2a) ala-sparsomycinl 2.0 206 163-248 5/84 (2h) ethyl-ala- 6.1 172 158-185 1/42 spars omyc in (2e) n-butyl-ala- 13.3 161 0/24 sparsomycin a. optimal dose was selected from an experiment with 3-5 dose levols. For selection following criteria were used: maximal TIC, no early toxic deaths (before clay 9) no weight loss exceeding 4 g at day b. number of the Long Term Survivors (cures) that survivjed the 60th day of experiment to the total number of animals used for experiments With each particular drug.

-43- TABLE 6 Antitumor activity of sparsomycin analogues on L1210 leukemia inoculated subcutaneously (106) on day 0. Treatment i.Nr. on day 1-7 Drug: optimal dose: mg/kg/i Median T/C Range:

W%

LTS/totala n-pentyl-sparsomycin 5.0 110/20 (2a) ala-sparsomycin 2.0 110/20 ethyl-ala- 6.9 139 122-156 0/42 sparsomycin TABLE 7 Antitumor activity of sparsomycin analogueG on P388 leukemia inoculated in CD2F1 mice ip (106) on day 0. Treatment ip on days 1-9 Drug: optimal dose: mg/kg/i m4edian T/C Range. LTS/totala Sparsomycin 0.125 90 0/24 n-pentyl-sparso~iyoin 5.0 161 146-175 0/48 benzyl-sparsomycin 6.0 133 0/24 n-octyl-sparsomycin 22.5 150 0/24 (2a) ala-sparsomryoin 2.9 153 136-170 0/48 (2h) ethyl-ala- 6.2 150 0/24 spar somyc in -44- TABLE 8 Antitumor activity of sparsomycin analogues on RC carcinoma inoculated 4p (106) cells on day 0. Treatment ip on days 1-9.

Drug: optimal dose: mg/kg/i Median T/C

W%

Range: LTS/totala sparsomycin 0, 125 100 0/24 n-pentyl-sparsomycin 3.9 414 227-600 8/48 benzyl-sparsomycin 3.4 149 139-158 0/48 n-octyl-sparsornycin 17.5 170 161-179 0/48 (2a) ala-sparsomycin 1,6 277 230-304 5/54 (2h) ethyl-ala- 4,1 197 0/24 sparsonyc in From the above it appears that (2a) ala-sparsoMycin and (2h) ethyl-ala-sparsomycin show higher antitumor activity than that seen with n-pentyl-sparsomyciti. (2h) ethyl-alasparsomycin is the only drug that is active on L1210 leukemia inoculated sc When the drug is administered iv.

This point to the exceptional properties of this compound.

Claims (4)

1. Sparsomycin (Sc-RS) compound having the general V formula P 0 1 13 CH where in R hY4~j4- C 1 6 alkyl, benzyl, iriazolemethyl, or

3-indo,,lmethyl; R= C1. 1 0 alkyl, or benzyl; R 3 =hydrogen; or RI and R 3 taken together represent (CH2)n wherein n 2. Sparsomycln Compound having the general formula 0 0 0 o! II13 CH R= C 1 4 alkyl, benzyl, imidazolemethyl, or R 2 C. alkyl, or benzyl; R3 hydrogen; or RI and R 3 taken together represent (CH2)n wherein n 46 and pharmaceutically acceptable salts thereof. 3. Pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, solvent, or excipiens and a therapeutically effective amount of a sparsomycin (Sc'Rs) compound as claimed in claim 1.

4. Pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, solvent, or excipiens and a therapeutically effective amount of a sparsomycin (Sc-Rs) compound as claimed in claim 2. A process for preparing a sparsomycin (Sc-Rs) compound having the general formula, 0 0 0 o o wherein R 2 and R 3 have the meanings defined in claims 1 or 2, comprising a series of reactions to convert 3 3 an Lamino acid having the general formula. e' fi S a O~ a B o j ,.I 3 1c into a compound having the general formula R 2 P, (12) R 3 and reacting said compound (12) with 6-methyl-uracil acrylic acid having the structural formula a 04 0 0 SLI 0 0 OH oH3 (14)

6. A process for preparing a sparsomycin (Sc-Rs) compound having the general formula i, n .II k"N. wherein R 1 I R2 and R 3 have the meanings defined in claims or 2, comprising the steps of reacting an L-amino acid methylester with di-tert. butylpyrocarbonate to obtain an N- (tert, buty oxycarbonyl) -t-amino acid methyl ester having the general formula "a /0; B0 N C 0 2 Me reacting said compound With a reducing agent to Obtain a compound having the general forMula BOO N OH I tosylating said compOUnd to obtain a compotund having the general formula Rt H,,1 BOO N N 7 reacting said compound with cesium O~rbona;te and thioacetic acid to obtain a thioester' having the general ormnula I 0 j~ 4 ~Yj ii reacting said thioester with acetic anhydride and chlorine to obtain the corresponding sulfinyl chloride, reacting said sulfinyl chloride with diazomethane to obtain the diastereoeric a-chloro sulfoxicles R 1 r BO N \Cnd N N t4 3R separating said diasterewmers and isolating the (Sc-R 5 a-hloro sulfoxide reacting same with a mercaptide having the general formula MSR 2 wherein M repreSents alkali metal to obtain a compound having the general formula 0 AR R i I deprotecting the amino group by treatmen t with trifluoroacetic acid to obtain a compound having the general foinula 1, 0 13 P. 4 0 and reacting same with 6-methyl-uracil acrylic acid, Dated this 29th day of January 1L988 STICH-TING TATHOLTEKE UNIVERSTTET Patent Attorneys for the Applicant F,B. RIQI CO,