SYNTHESIS AND EVALUATION OF ACYCLIC SUGAR NUCLEOSIDES

SYNTHESIS AND EVALUATION OF ACYCLIC SUGAR NUCLEOSIDES*

Derek Horton, David C. Baker, and Satish S. Kokrady

Department of Chemistry The Ohio State University

Columbus. Ohio 43210

INTRODUCTION

The object of this work was to synthesize nucleoside analogues having an acyclic sugar chain, to provide compounds for biological evaluation for their ability to mimic the natural cyclic sugar nucleosides, or to modify the solubility properties and transport-behavior of the attached bases in biological systems.

The following general synthetic approach was used.

R ' OH RCH-Br __t R -OR1 Br2 RfX( SEt I2 QEt

Path A rBth B

B s e p s e RCHOR'

I RCHSEt

R = - ( C H O A C ) ~ ~ O A C , "_ = 3 or 4, R' = a l e 1

The starting materials are the fully acetylated dialkyl dithioacetals of pentose and hexose sugars. These compounds react with bromine in inert solvents and undergo replacement of one alkylthio group to give an unstable monobromo derivative that is an acyclic analogue of the well-known glycosyl halides. This reaction was observed by Gauthier,' and has been quite extensively exploited by Weygand and coworkers,2 and by W ~ l f r o m . ~ These acyclic bromides can be coupled with nucleoside bases by standard procedures to give the corresponding acyclic-sugar nucleosides having an alkylthio group attached to C-1 of the sugar as well as the base. A variant of this synthesis involves the initial replacement of the bromine atom by an alkoxy group to give a monothiohemiacetal, which is then treated with bromine to replace the re- maining alkylthio group by bromine. The resultant halo ether is then coupled with the base as before, to give an acyclic-sugar nucleoside derivative that is the alkoxy analogue of the alkylthio derivative produced by the first route.

The structural characterization of these products poses a number of problems. For total characterization, it is necessary to establish definitively the point of cova- lent linkage of the sugar to the base and to determine the tautomeric form of the base: furthermore, a new asymmetric center is formed at C-1, and two isomeric products are, in principle, possible. Their separation and characterization, and the assignment of chirality at C-1 of these products, is not readily achieved by standard

-

* Supported by grants from the National Cancer Institute, grants no. CA 03232 and CA 15675. and from the National Institute of General Medical Sciences. grant no. GM-11976 (The Ohio State University Research Foundation Projects 759. 3980. and 1820).

131

132 Annals New York Academy of Sciences

chemical methods. Another point that is significant in understanding the behavior of these molecules concerns the conformation of the acyclic carbohydrate chain; if this chain is fully extended, the molecules would be topologically rather different from the natural cyclic sugar nucleosides, but if the chain readily adopts a folded conformation4 that would be approximately isosteric with the natural nucleosides, the possibility exists that such compounds might serve as effective antimetabolites competing with the normal nucleosides. The type of general structure synthesized is illustrated in the following depiction of an acyclic hexose chain in a hypothetical folded conformation, attached to an adenine residue. Also illustrated is an example of a so-called " homonucleoside" that differs from a normal nucleoside by the interposi- tion of a carbon atom between the glycofuranosyl ring and the nitrogeneous base. The latter type of nucleoside analogue can be prepared by routes very similar to those employed for the acyclic-sugar nucleoside analogues, namely, by starting with the dialkyl dithioacetal of a 2,Sanhydro sugar, which is subsequently coupled to the base by the same sequence (through an intermediate bromide) as is used for the acyclic-sugar nucleoside analogues. As will be shown later, this type of structure can also be generated from the acyclic-sugar nucleosides by effecting a chain-cyclization reaction to generate the 5-membered ring.

"Acyclic-sugar nuc l e 0 8 i de"

"Homonucleos ide"

Synthetic sequence

In the first synthesis of acyclic-sugar nucleosides, Wolfrom and coworkers' started with D-galactose and by way of the acetylated diethyl dithioacetal obtained two different crystalline products from coupling with adenine. These products were considered to be C-1 epimers; one was dextrorotatory and the other levorotatory, but the chirality at C-1 was not determined. Later, this same general procedure was used6 to couple a D-galactose chain to thymine.'

Applications with amino sugars' are illustrated in the following scheme, starting from the dithioacetal of 2-amino-2-deoxy-~-glucose protected by an N-2,4- dinitrophenyl group and by 0-acetyl groups; bromination followed by condensation with 6-benzamido-9-chloromercuripurine was used to afford the adenine nucleoside analogue, and replacement of the bromine by an alkoxy group, followed by subsequent chlorination and coupling again to the base, yielded the 1-alkoxy analogue of the acyclic-sugar adenine nucleoside, also.

Horton et al.: Acyclic Sugar Nucleosides 133

Further examples of these syntheses, in which six-carbon chains derived from D-galactose and D-glucose are coupled to thymine by way of a fusion reaction with the 2,4-bis(trimethylsilyl) derivative of thymine, are illustrated in the following scheme in which the products having alkylthio, alkoxy, and benzyloxy groups at C-1 were prepared.'.''

Uracil analogues were prepared by the same general route, as illustrated in the following scheme showing the preparation of a crystalline compound isolated as a

Br

CUR

HCOAc

I I I I I

HCObC

I

AcOCH -k

AcOCH

C H,OAc

R = -SEt R = -0Me R = -OCHZPh

- Me,S,O

Br

CHSEt I I I I I I

HCOAC

AcOCH

HCOAC

HCOAC

CHzOAc

I HCOAc

I AcOiH

I HCOAC

I I

HCOAc

CHzOAc

CUR

HCOAC I I

AcOiH

AcOCH

- I I I

HCOAC

CH20AC

R = - S E t

R = - 0 M e R = -OCHZPh

I I

HCOH

HOCH

I HCOH

I HCOH

I CHIOH

I I

HCOH

HOCH

I I

HOCH

HCOH

I C Y O H

R = -SEt R = -0Me R = -OCH2Ph


single epimer from a D-galactose precursor. In that study there were recorded the first examples involving pentose sugars (D-arabinose and ~-xylose) , which were con- verted into uracil nucleoside analogues and also into adenine derivatives.

B r I C H S E t I

I

I

I

I CH20AC

HCOAc

AcIXH

A c E H

HCOAc

I I C H S E t + I

I H E H

I H E H

I HCOH

CH20H

C H S E t I v HC OH I

I I

1 CH20Ac

HCOAc M e 3 S i 0 A c E H

AcOCH

HCOAc

one e p i m e r

157-80 208-9'

Condensation of the bromide from a D-XylOSe percursor with 6-acetamido-9- chloromercuriadenine gave an acetylated adenine nucleoside derivative that, despite its crystallinity, was shown by nmr spectroscopy to be an epimeric mixture, since the spectrum showed two different sets of signals for H-2 and H-8 of the purine residue. Separation of these epimers was not achieved.

Br C H S E t I

I

I

I CHzOAC

HCQAC

AcCCH

HCQAc

d H S E t I

2 steps HCQAc

+ <fi - A c ~ H

HCQAc igc1 I

CH,QAC

2 e p i m e r s

rap. 82-840

DISCUSSION

The principal thrust of the work described here is concerned with (1) the total structural identification of these acyclic-sugar nucleosides, (2) synthesis of the full stereochemical range of coupled products between selected bases and the D-pentOSeS to permit detailed examination of the conformations of the sugar chains and the influence of stereochemistry on the shape of these chains, and (3) the biological evaluation of these products to determine whether the attached sugar chain modifies the response of the base and whether the stereochemistry of the sugar chain has a significant effect on influencing the biological activity. Examples in the pyrimidine series are discussed first, followed by work conducted in the purine series.

Horton er al.: Acyclic Sugar Nucleosides 135

Acyclic-Sugar Pyrimidine Nucleosides

Starting from the acetylated diethyl dithioacetals of the D-pentOSeS, coupling by the fusion procedure with 2,4-bis(trimethylsilyoxy)pyrimidine gave the four corresponding acetylated uracil nucleoside analogues. Crystalline, dextrorotatory products were obtained in the D-arabinose and D-lyxose series; the product from D-ribose was crystalline and levorotatory, and the product from D-xylose was levorotatory and amorphous. The nmr spectra of these products showed only one set of signals for the protons at positions 5 and 6 of the uracil ring, indicating that they were single epimers at C-I.

0 0

I :() I () I f) 7

C H S E t C H S E t C H S E t C H S E t

0

I I I I AcOCH AcOCH HC OAc HC OAc

I I 1 I AcOCH HCOAc

I I I I I CH20AC

I I CH20AC J H 2 0 A c CH20AC

AcOCH HCOAC

HC OAC HCOAc HCOAc HCOAc

one epimer one epimer one epimer one epimer

140-2' 145-4' 102-50 amorphous

+ 1 p +140° -6 10 -1020

The tetraacetates were saponified to give the corresponding nonacetylated compounds, and these were again obtained as the corresponding single epimers.

k H S E t d H S E t 6 H S E t 6 H S E t I I I I

I I I I HC OH HOCH HC OH HOCH

I I I I HC OH HC OH HC OH HC OH

I I I I CH20H CHpOH CHzOH C H & H

HOCH HC OH HC OH HOCH

206-70 amorphous

al.1 a s s ingle epimers

After crystallization of the dextrorotatory tetraacetate in the D-arabinose series, the mother liquors from the reaction were found by nmr spectroscopy to contain the other C-1 epimer, which could be obtained as a levorotatory syrup that still contained some of the dextrorotatory epimer.

Similar syntheses in which 2,4-bis(trimethylsilyl)cytosine was used gave the corresponding cytosine analogues, as illustrated in the following sequence starting from D-xylose.


PH(SR)z HqOAc HqOAc Me3Si0

HFOAc HGOAc HCOA c AcOCH + Br2 - - t A c O c p

-0Ac C H ~ O A C c H 2 O A c

(+) epimer ( - ) epimer

In this example, both the diethyl and the diisobutyl dithioacetals were used as starting materials to afford the corresponding S-alkyl nucleoside analogues, and in each instance the acetylated products were isolated and separated as solids that were epimerically pure; one epimer was dextrorotatory and the other one levorotatory. Saponification of these products give the corresponding, crystalline, nonacetylated analogues.

In the D-arabinose series, the C-1 epimeric cytosine nucleoside analogues, one dextrorotatory and the other levorotatory, were obtained as chromatographically homogeneous, amorphous glasses. In the related D-lyxose and D-ribose series, these were obtained as epimeric mixtures that were not readily resolved, although partial separations could be achieved by chromatography on silica gel.

The next scheme illustrates extension of this synthesis to the 5-fluorouracil series by the same procedures that were used with uracil. The product ofcondensation with the D-arabinose derivative was obtained as a crystalline tetraacetate that was dextrorotatory and a single epimer, and deacetylation gave the corresponding dextrorotatory tetrol as an amorphous glass.I3

B r 2y mJy 4 I

I

I

I I

I I I

0% CHSEt

CHSEt I CHSEt I NH3, MeOH I I

AcOCH - AcOCH ,- HOCH HCOAc +

HCOAc HC6Ac HCOH

CGOA c HCOAc HCOH 1 CHzOH

I W O A c

88-90’ +80° (H20)

The acetylated acyclic-sugar nucleosides generally show weak molecular-ion peaks in their electron-impact mass spectra, together with a major high-mass ion resulting from cleavage of an alkylthio radical from the molecular ion. Other typical fragmentations include cleavage of acetate groups; fragmentation of the sugar chain

is particularly favored at the C-1-C-2 bond, giving rise to the BCHSalkyl ion. A ring-closure reaction linking 0 - 2 to C-5 in these products can, in principle, give

rise to the type of “ homonucleosides” mentioned earlier. Alternatively, such homonucleosides could be prepared by starting from the dialkyl dithioacetals of the corre-

+

Horton rt a/ . : Acyclic Sugar Nucleosides 137

sponding 2,5-anhydropentoses. Stereochemical factors play a major role in influencing reactions leading to the 2,Sanhydrides. The following scheme illustrates the behavior of dialkyl dithioacetals of the four D-pentOSeS on treatment a t low temperature with one mole of p-toluenesulfonyl chloride in pyridine.

H C ( S R l 2 H C ( S R )2 HC(SR12 H O ~ H

HC6H H C O H HCOH

TsCI I I TsCl 0 CH(SR12 H C O H .A/

I CSHSN I

I I CHaOH C H ~ O T S

p - 4 4

57 HCOH - I C5H5N H C O H I CH2OH HO OH

;-*

D-iE?? D-g&

The anticipated product of sulfonylation at the primary position is obtained in the D-arabinose series, but in the other three examples the sulfonate is not observed, and the 2.5-anhydride of the dithioacetal is formed instead. The factors controlling this reaction appear to be related to steric destabilization of the transition state for cyclization in the D-arabinose series, together with conformational considerations that facilitate the achievement of such bicyclic transition states in the other examples, particularly with the D-riho and D - X J ~ O configuration^.'^.' This differential reacti- vity can be used as the basis for synthesis of homonucleosides by two different routes.

Starting from the diethyl dithioacetal of D-XylOSe, Defaye and Machon16 prepared the corresponding acetylated 2,Sanhydride which, on bromination with subsequent addition of pyridine, gave the corresponding I-ethylthio-1-N-pyridinium

CH( SEt) , CH( S E t ) z HC I I OH T s C 1 , C&N- ~ c H ( s E t ) z AcpO 0 HCOH

HCCH 2 h, -10' CsH5N I 18 h, 25'

i") Br' N I

MeOH

12 h, 95' I I OAc OAc

128' (dec) I .."

175-7' -106'


I-

I CHSEt I

HOCH I

HfOH

HC OH 1 CHZOTS,

bromide; this was coupled with adenine to give the corresponding homonucleoside as a crystalline, levorotatory product. Alternatively, the 2,5-anhydride ring can be generated in the preformed acylic sugar nucleoside, as illustrated in the following example that compares the uracil nucleosides derived from D-XylOSe and D-arabinose."

n r

0 -3 & CHsEt TsC1, Cs&N

HC I I OH 2 18 h, h, -10' 25' - 1 a'HsE' HoFH

bH HC OH

I CH20H 1

XJ& precursor

0

0 0 6 H S n TsC1, C&N I

2 h, -10' HOCH 1 18 h, 25'

-- H?OH

HCOH

liH20H

arabino precursor

6AC

cycl izat ion of side-chain occurs

0

no cycl izat ion of sugar chain

Treatment of the arabinose derivative with one mole of p-toluenesulfonyl chloride in pyridine, followed by acetylation of the product, gave the acyclic-sugar derivative sulfonylated at 0-5. By contrast, the same procedure applied to the D - X Y ~ O derivative led to cyclization of the side chain and formation of the homonucleoside. Compari- son of this product with the corresponding analogue having 6-benzamidopurine as the base, obtained by the routeI6 employing a preformed anhydro sugar, showed very similar nmr parameters for the sugar portion in the two compounds, and mass-spectral comparison of the two products showed close parallels in the fragmentation modes.

The conformations of these acyclic-sugar nucleoside analogues have been explored in detail to determine whether the chains adopt extended or folded conformations." The idea of conformational folding of acyclic chains, to mimic the glycofuranosyl skeleton, has been advanced a number of times in the literature. For example, Coutsogeorgopoulos's has suggested that the side chain of chloramphen- icol is stabilized as a cyclic form through internal hydrogen-bonding to generate a structure that has at least a superficial similarity to a nucleoside in its molecular

The general conclusions from these studies are in line with the results of detailed investigations on various series of acyclic-sugar derivatives in s ~ l u t i o n , ~ and indicate that the planar, zigzag conformation of the acyclic chain is favored so long as this

topology.27


arrangement does not cause two substituents in 1,3-disposition to lie on the same side of the chain; if such a parallel 1,3-interaction would result, there is a rotation about a C-C bond of the chain to generate a “sickle” conformation in which the carbon chain is no longer in a fully extended orientation. This is illustrated here for acyclic pentose derivatives having two substituents a t C-1 ; the examples include the dithioacetal precursors, together with some of the corresponding acyclic-sugar nucleoside analogues.

OAc

D-arabino

Planar Zigzag

R‘ - R - SEt SEt S Ph S Ph SEt pyrimidine SCH2CHMr2 pyrimidine 0Mr OMc OAc H

AcO

H

R‘

I t may be seen that the D-arabino stereochemistry does not give rise, in the extended form, to an unfavorable interaction of substituents, and the nmr coupling- data are in full accord with this arrangement. By contrast, in the D-xylose series, the products d o not adopt the extended conformation, but instead exist as an equilibrium of “sickle” forms generated by C-C rotation to alleviate the destabilizing 1,3-interaction.

In the series of derivatives where D-arabinose was coupled with cytosine and uracil, it was possible to isolate both of the epirners at C-1 ; the following scheme tabulates the coupling data obtained” from each of these products. From the large


J I , values observed, i t can be inferred that H-1 and H-2 are antiparallel in each example; this fact is in accord with the idea that neither the pyrimidine residue nor the ethylthio group favor a disposition antiparallel to H-2, since this would generate a parallel interaction between a large C-1 substituent and the acetoxyl group at C-3. The epimer showing a J I . value of 9.8 Hz is evidently the derivative that is the more conformationally rigid ; this value is an extreme one, reflecting essentially complete conformational homogeneity. In contrast, the other epimer shows a somewhat lower value, indicative of a minor contribution by a rotameric form having H-1 and H-2 gauche-disposed.

=- D-arabino-1-( 2) me

A €I A AcO P

AcO H H OAc

D-arabinq-l-( I!)

The question of chiral assignment in these pyrimidine derivatives has not been established by a definitive method, although the nmr-spectral studies support the chiralities indicated in the preceding scheme. The products designated ( R ) were levorotatory at the sodium D line, whereas those designated ( S ) were dextrorotatory at this wavelength. The levorotatory epimers exhibited a positive Cotton effect near 275 nm that showed positive ellipticity, followed by a band near 240 nm showing a negative Cotton effect and negative ellipticity. The reverse behavior was observed for the dextrorotatory epimers. The fact that these assignments differ from those predicted from the Generalized Heterocycle RuleL9 may be attributed to the fact that the optical rotation at the sodium D line is influenced as a result of the band at 240 nm, which exerts a greater quantitative effect than the band at 275 nm that is presumably the one directly related to the configurational arrangement a t C-1. A definitive chiral assignment by x-ray crystallography in these pyrimidine derivatives has not yet been achieved.

The pyrimidine derivatives showed no significant inhibitory activity against Streptococcus,faecalis or against Escherichia coli (K-12); a 32% inhibition of growth of leukemia L-1210 cells at 1 x molar concentration was exhibited by the acyclic cytosine D-arabinose derivative, and about the same activity was observed with the uracil D-galactose analogue.20 Testing results in uiuo are not yet available.


Acyclic-Sugar Purine Nucleosides

Acyclic-sugar nucleosides of 6mercaptopurine were prepared from D-glucose and D-galactose precursors by the following sequence.

0r

CHSEl I I I I I I

HCOAC

ArOCH +

ArOCH

UC&

c n p c

<h I

HQCl

I I I I I I C H p C

CHSEt

HCOAC

AcOCH

AcOCH

HCOAC

I I I I I I

CHSEt

HCOAC

AcOCH

AcOCH

HCOAc

CI ipAc

CHSEt

I I

How I I C%W

- 1 nccu

HOCH

HCOn

189-190" g-galacto - Series -101' (H20)

<:fi <yJ &r Br

C H S E t I I

I I

I I I

I CH20Ac I

I

I I CHSEt &

I I H!OAC s c m H ~ A C MeOH HCOH

I I I

I I I I

CHSEt

HCOAc CHSEt

__I AcOCH - AcOCH HOCH

HCOAc

HCOAc AcOCH

H C W HCOAC

HCOAC HCOH

I HCOAc

HCOAc

cHzOAC CH20H CHzOAc

D-gluco Series - 222-224 -114" ( H 2 0 )

The initial condensation was conducted to attach 6-chloropurine to the sugar chain, and the chloro group was then replaced by sulfur through the action of thiourea. The protecting groups were then removed by use of methanolic ammonia to yield in each instance a crystalline, levorotatory nucleoside analogue.21 By analogy with known coupling reactions of 6-chloropurine, it may be inferred that the substitution is on position 9 of the purine ring, although this point is not established definitively by the mode of synthesis, nor is the tautomeric form of the base established. X-ray crystallographic studies22 on 6-mercaptopurine itself indicate that the isolated base exists as the thione tautomer. The chirality at C-1 of the sugar chain is not established, although the properties of the products indicated that they were single epimers and, for reasons advanced later, these products are considered to be most probably the 1 ( R ) epimers.

The chloro intermediates in the syntheses can also be isolated as the crystalline 0-deacetylated products, as depicted for the D-glucose example; whereas the crude product from the coupling reaction contained a small proportion of a second epimer, the crystalline product isolated was epimerically pure, strongly levorotatory, and


presumably of the same C-1 epimeric configuration as the foregoing 6-mercapto derivatives.

HCOAc

HFOAc HFOAc

A c O p

cH&c

IC&, MeOH H$OH

H ~ O H H$OH

HOCH

137-138' 179-180'

-105' ( C H C l s ) -1Wo (H20)

& 588, 590 (M'), 155 (M - 433)'

No detailed information as to the shape of the acyclic sugar-chain is yet available for these products, although physical data on acyclic-sugar derivatives having the D-galacro configuration indicate that the fully extended, planar zigzag conformation is favored, in contrast to the D-gluco analogues, where the extended form is destab- ilized and a folded structure is favored. This information lends credibility to the speculation that a nucleoside having the D-gUlactO side-chain may have this chain fully extended, as depicted in the following scheme, by contrast with the D-glUCO analogue, which might readily adopt a folded-chain arrangement similar to the topology of a natural nucleoside derivative. On the basis of this hypothesis, significant differences should be observed between the biological activity of the stereoisomers, whereas if the sugar chain is merely serving as a water-solubilizing carrier for the base (itself a known antimetabolite), there should be little difference in the biological activity of these two stereoisomers.

2 "LZ) "$q CHSEt I

HCOH I

OH I

HO ets

HOCH

HOCH

";i I CHSEt I

HCOH I HO HO

HOCH I

HCOH I

HCOH 1 CH20H CH20H

I no HCOH I

g - w Hypothetical Folded Conformations

?-sRlReto

FIGURE 1 illustrates that there are indeed significant differences in biological activity between the two derivatives. In the in oiuo leukemia L-1210 screen in the mouse, the D-gluco compound was active, showing a T/C ratio of 147 at a dose rate of 400 mg/kg, whereas the D-gUhCt0 compound was inactive and n o n t ~ x i c . ~ Similarly, in the inhibition screen against E. coli (K-12) the D-ghco compound showed 50% inhibition at 4 x molar. whereas a concentration of molar of the D-

Horton et ul.: Acyclic Sugar Nucleosides 143

CHSEt CHSEt I I

I I

I I

I I

I I

HCOH HCOH HOCH HOCH HCOH HOCH HCOH HCOH CH,OH CH,OH

Ornonim Yolorlty f o r 502 inhibition p0locto

StrrDtcooccus forco1is 4 x 16‘ 1 0- Oohrrlchlo K-12 4 10-0 10-3

%aukamla L-1210 cells lo-‘ lo-‘

~ e u k e m i o L-1210 (nouse) TIC TIC 141 100

(In vitro)

(In vlvo)

(4OOmQ/Kg) (2oomQ/KP)

FIGURE 1. Variations in biological activity of acyclic-sugar nucleosides as a function of stereochernistry.

galacro compound was not inhibitory.’” This observation suggested that the hypothesis of stereochemical influence was valid. and prompted us to synthesize the full range of u-pentose analogues for study of their biological behavior and to determine their conformations by nmr spectroscopy. It may be noted that other types of antitumor compounds derived from sugars have exhibited significant differences according to stereochemistry. For instance, the alditol derivative 1,6-bis(2-chloroethylamino)- 1,6-dideoxy-~-mannitol is a potent antitumor agent (“mannitol mustard,” “manno- mustine ”), whereas the D-glUCO analogue is inactive.24 Similarly, among a range of alditol a,odimethanesulfonates, only the 1,l-disubstituted L-threitol derivative and the 1,6-disubstituted D-mannitol derivative were found active; other derivatives having different stereochemical configurations were inactive.25

The following scheme illustrates the synthesis of acyclic-sugar nucleosides having the D-pentoses attached to 6-mercaptopurine. The 6-chloro derivatives were obtained in each example as gums that were mixtures of the C-l’epimers except for the D-aruhino derivative, which was a single epimer. At the succeeding step, the acetylated 6-mercaptopurine derivatives were isolated crystalline in yields of 50- 70‘Y0, and the crystalline products in the D-riho, D-arabino, and ~ - x y / o series were single epimers in each instance. whereas the n-lrxo compound. although cryst a I I ‘ me. was a mixture of epimers. Optical rotatory data on these derivatives are recorded in TABLE 1, where it is seen that the D-ribo and D-arabino derivatives are strongly dextrorotatory, whereas the D-XY~O derivative is strongly levorotatory ; the lyxo derivative has an intermediate value. By application of the Generalized Heterocycle Rule.’” it may be inferred that the dextrorotatory compounds have the I - (R) configuration, whereas the levorotatory D - X J ~ O compound has the I-(S) configuration; the D-/~.Yo compound is shown by nmr spectroscopy to be a mixture of C-1 epimers.


C H ( S E t ) Z I

(CHOAC) I + CHzOAc

a-d

Br I CH - PEt I

(CHOAc13 I CHZOAc

a- d

kCl CHSEt I

( C H O A C ) ~ I CHZOAc

a-d a-d

Synthesis of the l-S-ethyl-l-(1,6-dihydro- - 6-thioxopurin-g-yl) -1-thio +-pentito - 1s

These assignments are further supported by optical rotatory dispersion studies, as shown in FIGURE 2, where it may be seen that the D-ribo and D-arabino compounds display positive optical rotatory dispersion curves of large amplitude in the 250-400-nm region, and the D - X J ~ O compound shows an equally large, negative curve. As expected, the 0.r.d. spectrum of the D-/YXO product showed the result of the presence of a mixture of C-1 epimers.

A decisive proof of the stereochemistry at C-1 was provided by an x-ray crystal- structure analysis26 of the D-arabino derivative (FIGURE 3), conducted in the crystallography laboratory of the Institute for Natural Products at Gif-sur-Yvette, France.

The relative stereochemistry determined by the crystallographic analysis, in con- junction with the known absolute stereochemistry of the D-arabinose precursor, showed that the chirality at C-1 is 1-(R), in agreement with the configuration presumed on the basis of optical rotatory dispersion data. The crystallographic analysis also establishes decisively that the sugar chain is attached at position 9 of the heterocycle, and the length of the linkage between C-6 of the heterocycle and the sulfur atom (1.67 A) indicates that the heterocycle is in the thione tautomeric form.

TABLE 1 OPTICAL ROTATORY DATA FOR 2,3,4,5-TETRA-0-ACETYL-

1 &ETHYL- 1 -( 1,6-DIHY DRO-6-THIOXOPURIN-8-Y L)- 1 - THIO-D-PENTITOLS

[a];’ in CHCI, Configuration Compound (degrees) Indicated at C-1

D-ribo + 185 R D-UrUhitlO + 131 R D-XYlO - 179 S D-lYX0 f 41 R , S


FIGURE 2. Optical rotatory dispersion spectra of l-S-ethyl-l-( 1,6-dihydro-6-thioxopurin-9- yl)-l-thio-~-pentito1 tetraacetates.

FIGURE 3. The x-ray crystal structure of ( IR)-2.3.4.5-tetra-0-acetyl-l-S-ethyl-l-( 1.6-dihydro-6-t hioxopurin-9-yl)-l-thio-~-arabinitol.*~


Furthermore, the conformation in the solid state is found to be very close to a planar, zigzag arrangement of the carbon atoms of the sugar chain, with the ethylthio group in the extended disposition and the sulfur atom antiparallel to C-3, and the heterocycle in a gauche disposition; the acetoxyl group at C-5 is also gauche-disposed. A very similar conformation for this molecule in chloroform solution is indicated by its nmr spectrum, as shown in FIGURE 4.

FIGURE 4. Partial nmr spectrum (Me,SO-d,) of (lR)-2,3,4,5-tetra-O-acetyl-I-S-ethyl-l-( 1.6- dihydro-6-thioxopurin-9-y1)-1 -thio-~-arabinitol.

From the observed spin-coupling data between the protons along the backbone chain, it is clear that the ethylthio group occupies the extended orientation and the base is in a gauche disposition, and the carbon atoms of the sugar chain are in an extended, planar zigzag arrangement. The spin-coupling values between H-4 and the two protons at C-5 suggest that the molecule is probably a rotameric mixture of that form having the 5-acetoxyl group in the extended orientation (antiparallel to C-3) together with the form having the 5-acetoxyl group antiparallel to H-4.

In contrast to the nmr data for the D-arabino compound, the D-ribo derivative shows couplings that, as anticipated, support a nonextended conformation of the sugar chain. The data determined from the spectrum are best accommodated by postulating a mixture of two sickle conformers in equilibrium (FIGURE 5), with the major one being derived from the extended form by rotation about C-3-C-4, and the minor one being generated by rotation about C-2-C-3.

Deacetylation of the four acetates by use of butylamine gave the corresponding free tetrols. In bacterial-inhibition studiesz0 against E. coli (K-12), notable differences in activity were observed between the stereoisomers. Some of the test results are given in TABLE 2. The D-ribo compound is seen to be an order of magnitude more active than the other stereoisomers in inhibiting growth of this organism, and similar differences are also observed in the assay against E. coli B and against Streptococcus faecalis. Therefore, it is evident that, as in the example with the six-carbon sugar

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TABLE 2 fn Vitro BIOLOGICAL ASSAYS FOR I-S-ETHYL-1-( 1,6-DIHYDRO-6-

THIOXOPURIN -9-YL)- 1 -THIO-D-PENTITOLS * Molar Concentration for 50% Inhibition

Configuration E . coli K-12 E . coli B S . faecalis L-1210t rib0 8 x lo-’ 8 x lo-’ 6 x lo-’ 8 x lo-’ arabino 8 x 8 x 2 x > xylo 2 10-4 2 10-4 5 x 10-4 > 10-4 lyxo I 10-4 1 10-4 3 x 10-4 > 10-4

* Data courtesy of Dr. A. Bloch. Values checked in duplicate. t Cell culture.

chain, the stereochemistry of the side chain plays a considerable role in influencing the biological activity of the compound. Conformational analysis of the unsubstituted compounds is more difficult than with the substituted ones, but inspection of the nmr data for the D-rib0 derivative in water solution shows couplings that accord with a conformational mixture very similar to that observed for the acetates, as shown in FIGURE 6.

no \-

Major conformer in Cc,DSN Minor conformer

FIGURE 6. (1R)-l-Deoxy-l-S-ethyl-l-(1,6dihydro-6-thioxopurin-9-yl)-l-th~o-~-ribitol. J , . , 2 , = 2 . 1 ; J , . . , . = ~ . O ; J , , , , . = ~ . ~ H Z .

Further work on this problem is concerned with detailed comparative biological evaluation of all of the products, with chemical correlation of the nucleoside derivatives with the established stereochemical reference determined crystallographically, with the detailed conformational analysis of the unsubstituted products, and with the unequivocal assignment of chirality to the acyclic pyrimidine nucleoside analogues.

SUMMARY

Acylated aldose dialkyl dithioacetals with bromine undergo replacement of one alkylthio group by bromine. These unstable bromides react, as by fusion with 2,4- bis(trimethylsilyloxy)pyrirnidine, to give acylated I-(pyrimidin-I-yl) derivatives that


upon saponification afford acyclic sugar nucleoside analogues, some as separable mixtures of I-epimers. Systematic stereochemical variants have been conducted. Pmr conformational studies show that the sugar chain is extended in certain examples, whereas others favor folded (“sickle”) conformations, in line with a general rationale developed for acyclic-sugar derivatives. Condensation of the bromides with purines gives 9-substituted acyclic-sugar nucleoside analogues; synthesized systematically for various series, these include the D-pentoses in combination with 6-mercaptopurine. I n oirro and in ciuo biological activities vary according to stereochemistry of the sugar. The position of substitution of the sugar chain, the chirality a t C-l’, and the tautomeric form of the heterocycle, were established by x-ray crystallography of the product from D-arabinose and 6-mercaptopurine. The x-ray data permit correlation of C-1 chirality throughout the series and pmr data indicate the favored conformations.

REFERENCES I. GAUTHIER, B. 1954. Ann. Pharm. Fr. 12: 281-285. 2. WEYGAND. F., H. ZIEMANN & H. J. BESTMANN. 1958. Chem. Ber. 91: 2534-2537. 3. WOLFROM, M. L. 1972. In The Carbohydrates: Chemistry and Biochemistry. W. Pigman and

4. HORTON, D. & J. D. WANDER. 1974. J. Org. Chem. 39: 1859-1863; HORTON. D., P. L.

5. WOLFROM. M. L., A. B. FOSTER, P. MCWAIN, W. VON BEBENBERG & A. THOMPSON. 1961. J.

6. Woi.kRoM. M. L.. P. MCWAIN & A. THOMPSON. 1962. J. Org. Chem. 27: 3549-3551. 7. WOLFROM. M. L.. W. VON BEBENBERG. R. PAGNUCCO & P. MCWAIN. 1965. J. Org. Chem.

8. WOLFROM, M. L., H. G. GARG & D. HORTON. 1965. J. Org. Chem. 29: 3280-3283; 1966.

9. WOLFROM. M. L., H. B. BHAT, P. MCWAIN & D. HORTON. 1972. Carbohyd. Res. 23:

10. WOLFROM. M. L. & P. J. CONIGLIARO. 1971. Carbohyd. Res. 20: 369-374. 1 I . HORTON. D. & S. S. KOKRADY. 1972. Carbohyd. Res. 24: 333-342. 12. KOKRADY. S. S. 1972. Ph.D. Thesis. The Ohio State University, Columbus, Ohio; DEFAYE.

13. HOKI‘ON, D. & R. A. MARKOVS. 1973. Abstr. Papers Amer. Chem. SOC. Meeting. 166:

14. DttAYE, J. & D. HORTON. 1970. Carbohyd. Res. 14: 128-132. 15. Diir;AYa. J.. D. HORTON & M. MUESSER. 1974. Abstr. Papers Amer. Chem. SOC. Meeting 168:

16. DEFAYE. J. & Z. MACHON. 1972. Carbohyd. Res. 24: 235-245. 17. HORTON. D.. S. S. KOKRADY & J. D. WANDER. T o be published. 18. COUTSOGEORGOPOULOS, C. 1967. Biochem. Biophys. Res. Commun. 27: 46-52. 19. El. KHAixM. H. & 2. M. EL SHAFEI. 1963. Tetrahedron Lett. 27: 1887-1889. 20. BLOCH. A. Personal communication. 21. WOLFROM, M. L., P. MCWAIN. H. B. BHAT & D. HORTON. 1972. Carbohyd. Res. 23:

22. SLETTEN. E.. J. SLETTEN & L. H. JENSEN. 1969. Acta Crystallogr. 825: 1330-1338. 23. WWD. H. B. Personal communication. NCI cancer-screening data. 24. VARGHA, L., L. TOLDY, 0. FEHBR & S. LENDVAL. 1957. J. Chem. SOC. 805-809; compare L.

25. FEIT, P. W. 1961. Tetrahedron Lett. 20: 716-717; WHITE, F. R. 1962. Cancer Chemotherapy Rept. 24: 95-97; HADDOW, A.. G. M. TIMMIS & S. S. BROWN. 1958. Nature 182: 11641 165: WHITE. F. R. 1962. Cancer Chemotherapy Rept. 23: 71-79.

26. BAKER. D., A. DUCRUIX, D. HORTON & C. PASCARD-BILLY. 1974. Chem. Commun. 729- 730. 27. JARDETZKY, 0. 1963. J. Biol. Chem. 238: 2498-2508.

D. Horton, Eds. IA: 355-390. Academic Press. New York, N.Y.

DLIRETI‘E & J. D. WANDER. 1973. Ann. N.Y. Acad. Sci. 222: 884-914.

Org. Chem. 26: 3095-3097.

30: 2732-2735.

30: 1096- 1098.

289 295.

J. D., D. HORTON, S. S. KOKRADY & Z. MACHON. 1975. T o be published.

CARB-19.

CARB-I.

296-300.

VARGHA & T. HORVATH. 1959. Nature 183: 394395.


DISCUSS~ON OF THE PAPER

DR. VORBRUGGEN: I could imagine that if you reacted the dithioacetal directly with tin tetrachloride and a silylated uracil or cytosine, you could save one or two steps and get directly to those products. The silylated pyrimidine is completely stable; you have just to treat it with tin tetrachloride or with titanium tetrachloride, which is a bit rougher.

DR. HORTON: That is a possibility. We did make some studies with titanium tetrachloride, and obtained rather complex mixtures. But the tin tetrachloride looks promising.

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SYNTHESIS AND EVALUATION OF ACYCLIC SUGAR NUCLEOSIDES

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