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SYNTHESIS OF NUCLEOSIDE MONO-AND DIALDEHYDES AS ANTIVIRAL AGENTS

Annual Report

John P. Neenan, Ph.D.

0)

15 December 1987

U.S.ARM MEDCAL Supported by

U.S.ARM MEDCALRESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMDl7-86-C-6002

Rochester Institute of TechnologyRochester, 14ev York 14623

Distribution~ to US Government Agencies only; Pr~oprietaryInformation, January 4, 1988. Other requests for this documentmust be referred to Comander, US Army Medical Research andDevelopment Command. ATTN: SGRD-RMI-S, Fort Detrick, Fredstrick,Maryland 21701-5012.

The findings in thi.s report are not to be construedas an official Department of the Army position unleass

so designated by other authorized documents

~4D""1C

SAUG 1 51988~

SECURITY CLASIIAINO TmilS PAGE

REPORT DOCUMENTATION PAGEIs. REPORT SECURITY CLASSIFICATI ON lb. RESTRICTIVE MARKIUNG

Unclassified _______________________

2&. SECURITY CLASSIFICATION AUThORITY 3. DISTRIBUTION JAVAILAWSFY OF REPORT

26. DECLASSIFICATIONIOOWNGRADING SCHEDULE See cover.

4. PERFORMING ORGANIZATION PEPORT NUMBER(S) S. MONITORING ORGANIZATION REPORT NUMBER(S)

6.. NAME Of PERFORMING ORGANIZATION 6b. OFFICE SYMIBOL. 7a. NAME OF MONITOWiNG ORAANiZATI0N

Rocherter Institute of(I pdcb)

TechnologyJ6C. ADDRESS (Cd)'. State. and ZIP Code) 7b. ADCRESS (City, State. OutZIP Co*.)

Rochester, NY L4623

I&. NAME OF FUNDiNGiSPNSORING O b. OFFICE SYMBOL 9. PROCUREMENT INSTRUA~tr IDENTIFICATION NUMBERORGANIZATION U.*S.* Army Mied icali (if Applicable)

esearch & Development Command ________ Contract No. DAND7-86-C-6002&C. ADDRESS (Ciy. State. and ZIP Code) 10 SOURCE OF FUNDING NUMBIERS

Fort Detrick PROGRAM PROJECT TASK IWORK UNIT

Frederick, ND 21701-5012 ELEM6ENT NO. INO O. I ACCESSION NO.

It. TITLE (include Securrty Classoicationj. 670AA

Synthesis of Nucleoside Mono- and Dialdehydes as Antiviral Agents (U)

12. PERSONAL AUTHOR(S)John P. Neenan, Ph.D. __________

13a. TYPE OF REPORT 136. TIME COVERED 14. DOATE OF REPORT (YMJW,?tAay) 15. PAGE COUNT

Annual FROM ll-15-86TO11-l4-81 1987 December 15IL SUiPLIMINTARY NOTATION

17. COSATI CODES 1S. SUBJECT TERMS (continue an reverse if neceuuuv and ode-tf by block numbmFIELD GROUP SUB-GROUP

06 101NUCLEOS IDE DIALDEHYDES06 010 ANTIVIRAL AGENTS

19. ABSTRACT (Continue on reverse of neceiwy and tdvntify by block number)

rTwo compounds, 4' ,5'-unesturated adenosine-2 ,3'-dialdehyde and tuiercidin-2 ,31-dialde-hyde were submitted during the reporting period. Work on the synthesis of adenomine-2-monoaldehyde, adenosin*-3V-sonoaldehyde and 5'-deoxyadenosine-2',V-dialdehyde is stillin progress. As a group th" nucleoside dialdehydes thus far submitted have shown broadspectrum activity against wainy of the viruses in the screening system, and some, likeguanosine diaLdehyde, have shown remarkably Low cytotoxicity. Even dLaldehydes thatcannot become phosphorylated, which is required for the antiviral activity of all clinicallyused nucLeomide antivirals, shoved antiviral activity. For example 5'-fluoroV5-deoxy-admnosine-2',3'-dialdehyde showed good acti'Vity against yellow fever and 4',5'-unsaturaitedadenosin*-2',3'-diLsdehyde ahowed excellent activity against vesicular stomatitis virus.

20. DISTRIBUTION /AVAILABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATIONOUNCLASSIFIED/U.NLIMITEO 03 SAME AS RPT 03 DTIC LISERS UNCLASSIFIED

22a. NAME Of RESPONSIBLE INDIVIDUAL 122b TELEPHONE (INc~w AfyaCuaj 22c. OFFICE SYMBOL

*Ifrrfnfa M. H1111r I (301) 6-637325 SCRD-RMt-S00 Form 1473. JUN 84Prewo" # I01 s oni obsolete. SECuNFY CLASSIFICATION -OF THIS PAGE

Summary

Two compounds, 4',5'-unsaturated adenosine-2',3'-dialdehyde and tuber-cidin-2'3'-dialdehyde were submitted during the reporting period.Work on the synthesis of adenosine-2'-monoaldehyde, adenosine-3'-mono-aldehyde and 5'-deoxyadenosine-2',3'-dialdehyde is still in progress.As a group the nucleoside dialdehydes thus far submitted have shownbroad spectrum activity against many of the viruses in the ocreeningsystem, and som*, like guanosine dialdehyde, have shown remarkablylow cytotoxicity. Even dialdehydes that cannot become phosphorated,which is required for the antiviral activity of all clinically usednucleoside antivirals, showed antiviral activity. For example 5'-fluoro- A.5'-deoxyadenosine-2',3'-dialdehyde showed good activity against yellow b

fever and 4',5'-unsaturated adenosine-2',3'-dialdehyde showed excellentactivity against vesic lar stomatitis virus.

F';;

Accession oro

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Foreword

Citations of commerical organizations and trade names in this reportdo not constitute an official Department of the Army endorsement orapproval of the products or services of these organizations.

-2-,

7i

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Table of Contents

Page

Suary 1Foreword 2Technical Presentation 4

Chemistry 4Table I. Compounds Submitted During Reporing Period 5Biological Results 8Conclusions and Recomendations 8Tables II-XI. Antiviral Activity of Compounds 9-15

Submitted During Contract Period To DateExperimental Section 16

Literature Cited 19List of Personnel Supported by the Contract 20Distribution List 21

.. Fop

NV.

S.%

-3-

Technical Presentation

Background

Last contract year, compounds 1-5,7 and 9 were prepared by periodicacid oxidation of: inosine, 6-methyluercaptopurine ribosid!, thymineriboside, guanosine 5'-fluoro-5'-deoxyadenosine (compound 6), 8-bromo-adenosine and N6 , N6-dimethylaninopurine riboside, respectively. Thesynthesis of these compounds, which were submitted for antiviral screeningduring the last contract year, were described in the last annual report.Work toward the synthesis of the 4',51-unsaturated derivative (8) ofadenosine dialdehyde, as well as the unsuccessful attempt to synthesizethe dialdehyde derivative (10) of formycin A was also described inthe last annual report, which contains the structures of compounds1-10.

This annual report describes the successful completion of thesynthesis of compound 8 as well as the synthesis of tubercidin-2',3'-dialdehyde (11). Compound 8 and 11 were the only two compounds submittedduring this reporting period. This report also describes work in progresstoward the synthesis of adenosine-2'-sonoaldehyde, adenosine-3-sonoaldehyde,and additional 5'-deoxyadenosine-2',3'-dialdehyde (AVS #1214). Biologicalresults obtained on all compounds thus far submitted for screeningis also presented and evaluated in this report.

Chemistry

4',5'-Unsaturated adenosiae-2',3'-dialdehyde (8) was preparedby modification of the method of Grant and Lerne 1 . Periodate oxidstionof tubercidin by the method of Dvonch et al. 2 gave tubercidin-2',3'-dialdehyde (11). Details on the synthesis and characterization ofcompounds 8 and 11 (Table i) is presented in the Experimental Section.

Work towards the synthesis of adenosine monoaldehydes is describedin Scheme I. Periodate oxidation of 5-0-trityladenosine (Sigmas ChemicalCo.), compound 12 gave dialdehyde 13, which was then reduced to compound14 with sodium trimethoxyborohydride. Adduct 14 was treated with excesstrttyl chloride in an attempt to prepare the ditrityl derivatives 15and 16. This reaction, however, produced the tritritylated derivative17 instead of the desired intermediates. The structure of 17 was positivelyidentified by NHR. We are confident that more controlled tritylationwill afford the desired 15 and 16, and this reaction is currently underway.Pfitziter-Moffatt oxidation ooi15 and 16 should afford blocked monoAldehydes18 and 19 which should readily be able to be deblocked to afford targetmonoaldthydes 20 and 21.

Work towards preparation of more AVS #1214 is described In SchemeIU. Treatment of adenosine (22) with thionyl chloride in acetonitrilefollowed by aqueous sodium carbonate hydrolysis of the sulfite intermediate(not shown) has thus far afforded approx. 5 grams of 5'-chloro-5'-deoxyadenosine(23) and work Is in p- :ress to prepare still more of this derivativewhich will be dechloria.ated to 24, which in turn will be oxidized totarget 25.

-4-

•pp

Table 1. CompounJs Submitted During reporting Period

NH 2•N NH2

H IIICH HC CH0 0O00

Coablu d Code No. Ave No. Namero

8 OP-1-143 2906 4',5'-Unsaturated adenosin*-2',3'-dialdehyde

11 MTNI-1-7 2907 luberctdtn-2',3'-dialdehyde"

00 00

__ fI

Compund ode o. AS No Nam

Scheme I

TOTrOCH 2 A TrOF2 TCH2 A

H5106 Na(OMO)3 BH -4-p a-HC CH H2 C H2

HO CH ili 1 10 0 HO CH

12 13 14TrCI ~excess

TrCl

Trý2ATrOý2A 0h~2

H2C C42 + H29C H2 H2C 0421r rlHO u~r TrO OTr

acetic anhydride 1

A"

H2C 04 HC?2

TrO 0 0OTris 19

I H~rfl-OAc IA *adenineItTr trityl

H2C 04 HC CH2

20 21

Scheme 11

22 V

JSOCI2 /aceton-trile

CICH2A

H-O. CH23

tributyltin hydrid./THF

CH3 A

24O

JH5b0 6 *

00

25

c7

Biological Results.

Antiviral screening data on all compounds submitted during thecontract period are presented in Tables II-XI. Salient results includethe following encouraging findings. 0

1. Compound 8, which cannot become phosphorylated intracellulary, %showed a 3.5-fold increase in therapeutic index and a 30-fold decreasein cytotoxicity against vesicular stomatitis virus (VSV) than adenosinedialdehyde (AVS #1160), vhtch, at least theoretically, can becomephosphorylated, and which previously was the most active compoundagainst VSV An the entire screening system.

2. Inosine dialdehyde (1), which was virtually inactive in vitro,did show in wivo activity against Crimean-Congo hemorrhagic feverin mice.

3. j'-Fluoro-5'-deoxyadenosine dialdehyde (5) showed good activit?against yellow fever while the parent nucleoside 6 did not. Thisindicates that the dialdahyde groups are crucial to the antiviralactivity of compound S. Thymine riboside dialdehyde 3 was alsoactive against yellow fever.

4. In one test, compound 7 showed significant activity against theAIDS virus (HIV), but this result will have to be confirned.

S. As a class the nucleoside dialdehydes thus far submitted both prior

to and during the contract period have shown broad spectrum activityagainst most of the viruses in thz screening system, and some, ,q.like guanosino dialdehyde (4) showed remarkably low cytotoxicity.

Conclusions and recommendations.

Plans and recomrtdauions for the current third contract year includethe followingt

I. Synthetic work described in Scheme I and II is still in progress.

2. 5'-Deoxyadenosine dialdehyde (25) will be converted to monoaldehydesfollowing a synthetic similar to Scheme I.

3. Another attempt will be made to synthesize formycin A dialdehyde(10).

4. Work will com ence on efforts to prepare V'-smino-5'-deoxyadouosine,diildehyde and otber remaining target ccmpcunds listed in the contractproposal.

S. Thymine r~boside dialdehyde (1) is somewhat similar in structureto AZT, was active againit yellow fever and should " screenedagainst HIV as soon as possible.

6. Other future carget compounds will be described in a forthcomingcontract renewal proposal.

Table II. Antiviral Activity of Inosine-2',3'-dialdehyde (Compound 1, -

AVS f 1915)

IN VITRO SCREEN [ug/all

VIR VR VR+ ID50 CELL MTC T! TI+ ,PT 0.4 220.00 PK2 32.00 0.1VSV NOT ACT VERO <250.00 NOT ACTVEE NOT ACT VERO <250.00 NOT ACTRVF NOT ACT VERO <250.00 NOT ACTYF KOT ACT MK2 <250.00 NOT ACTPIC NOT ACT VERO < 50.00 NOT ACTPIC NOT ACT VERO <250.00 NOT ACTVSV 0.0 1.0 NOT ACT L929 100.00 0.0 3.4 IAD2 >0.2 0.5 31.95 HEP2 <100.00 <3.1. 2.4VV 0.2 0.8 320.00 VERO 320.00 1.0 2.8PT >0.0 0.5 NOT ACT VERO >320.00 >0.0 4.6JBE >0.0 0.9 NOT Act VERO >320.00 >0.0 2.2SFS 0.3 0.7 26.19 VERO 32.00 1.2 4.6YF 0.1 0.8 NOT ACT VERO 100.00 0.0 1.4PT 0.0 0.4 NOT ACT VERO j20.00 0.0 4.6HIV 0.0 2.2 NOT ACT ATH8 32.00 0.0 >320.0

S

IN VIVO SCREEN (mg/kgj

VIR HST vR VR+ DOSE 'TC RTE Dcch mus 2.0 1.4 50 .s >50 IP 1 rcch -Us 0.8 2.7 50 mS >50 IP 1

Table III. Antiviral Activity of 6-HethylmercaptopurineRiboside-2',3'-dialdehyde (Compound 2, AVS 01970)

IN VITRO SCREEN [us/mal

VIR VR VR+ ID50 CELL MTC TI TI+PT 0.1 24.00 HK2 3.20 0.1RVF NOT ACT VLRO 25.00 NOT ACTVSV 0.2 1.0 17.88 L929 32.00 1.8 3.4AD2 MI. 0.5 SIOT ACT HEP2 <10.00 0.0 2.4 .,VV 0.0 0.8 NOT ACT VERO 32.00 0.0 2.8

PT >0.0 0.5 NOT ACT VERO >320.00 0.0 2.8JBE 0.1 0.8 32.00 VERO 32.00 1.0 1.1YF 0.0 0.6 NOT ACT VERO >320.00 0.0 1.8SS >0.0 0.8 NOT ACT VERO >320.00 0.0 8.5PT 0.0 0.4 NOT ACT VERO >320.00 >0.0 A.6

0

-9-

Table IV. Antiviral Activity of .Thymine Riboside-2't31-dialdehyde(Compound 3, AVS #1976)

IN VITRO SCREEN [ug/all

VIR VR VR+ ID50 CELL HTC TI T1+VSV 0.6 100.00 L929 100.00 1.0RVF NOT ACT VERO 50.00 NOT ACTAD2 0.2 NOT ACT HEP2 32.00 NOT ACTSF >0.8 16.43 VERO >100.00 >6.1PIC 67.00 VERO >100.00 >1.5YF 0.9 18.05 VERO 320.00 17.7JE >0.3 70.92 VERO >100.00 >1.4PT 0.6 45.00 M12 3.20 0.1PT 0.7 25.00 MK2 1.00 0.0PTVB 0.4 85.00 PK2 10.00 0.1YF 0.1 0.6 NOT ACT VERO 3.20 0.0 0.2PT 0.0 0.4 NOT ACT VERO >320.00 >0.0 4.6VER 0.0 0.3 NOT ACT VERO 100.00 0.') 1.2

Table V. Antiviral Activity of Guanosine-2',3'-dialdehyde(Compound 4, AVS #1211)

IN VITRO SCREEN lug/all

VIR VR VR+ ID50 CELL MTC TI TI+

RVF NOT ACT VERO >562.40 NOT ACTVEZ NOT ACT VERO >562.40 NOT ACTYF <562.40 M12 >562.40 >1.0VSV 73.43 VERO >562.40 >7.7PIC NOT ACT VERO >562.40 NOT ACTPIC NOT ACT VERO 568.00 NOT ACTRVF NOT ACT VERO 568.00 NOT ACTVER NOT ACT VERO 568.00 NOT ACTVSV 0.26 VERO 568.00 7.7YP 2.00 P, 2 568.00 1.0AD2 >0.4 0.8 4.91 HEP2 <32.00 <6.5 2.7VW 0.5 1.0 62.11 VERO 320.00 5.2 2.8PT >0.0 0.5 NOT ACT VERO >320.00 >0.0 4.6YF 0.0 0.7 NOT ACT VERO 32.00 0.0 0.35Sy 0.4 l,O 13.27 VERO 32.00 2.4 18.6JBg >0.1 1.0 NOT ACT VERO 320.00 >0.0 6.4PT 0.1 0.4 253.49 VERO 320.00 1.3 1.1

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Table VI. Antiviral Activity of 3'-Fluoro-5'deoxyadenosine-2'.3V-dialdehyde (Compound 5, AVS #2275)

IN VITRO SCREEN lug/all

VIR VR VR+ ID50 CELL RTC TI 71+

RVF NOT ACT VERO <250.00 NOT ACTAD2 0.5 57.50 HEP2 100.00 1.7VV 0.7 14.81 VERO 100.00 6.8VSV 0.1 31.00 L929 10.00 0.3SF 0.7 13.55 VERO 100.00 7.4FeLV 0.1 NOT A,,i 81C 10.00 0.0YF 1.1 13.13 VFRO 320.00 24.4HIV 0.3 1.4 NOT ACT ATHS 10.00 0.0 >32.0JE 0.2 61.68 VERO 100.00 1.6Hiv 0.0 NOT ACT ATH8 32.00 0.0YF 0.2 0.6 79.56 VERO 10.00 0.1 0.2

Table VII. Antiviral Activity of 5'-Fluoro-5'deoxyadenosineýCompound 6, AVS #2273).

IN VITRO SCREEN lug/mll

VIR VR VR+ ID50 CELL HTC TI TI+

VSV 0.0 NOT ACT L929 100.00 0.0AD2 M. NOT ACT HEP2 <100.00 0.0VV 0.1 NOT ACT VERO 100.00 0.0¥F 0.3 98.43 VERO 10.00 0.1SF 0.0 NOT ACT VERO 100.00 0.0JR 0.0 NOT ACT VERO 10.00 0.0

-11-

Table VIII. Antiviral Activity of 8-Bromoadenosine-2',3'-dialdehyde

(Compound 7. AVS #2274)

IN VITRO SCREEN (ug/al]

VIR VR VR+ ID50 CELL MrC TI TI+RVF 132.47 VERO <250.00 1.90AD2 >0.1 NOT ACT HEP2 <100.00 0.0VV 0.1 NO• ACT VERO 32.00 0.0VSV 0.1 NOT ACT L929 32.00 0.0SF 0.7 15.88 VERO 100.00 6.3YF 0.4 22.35 VERO 3.20 0.1FeLV 0.0 NOT ACT 81C 10.00 0.0HIV 0.4 1.2 0.32 ATH8 3.20 10.0 >32.0JE 0.3 91.75 VERO 100.00 1.1HIV >0.0 NOT ACT ATH8 <100.00 (0.0

Table IX. Antiviral Activity of 4',S'-Unsaturated Adenosine-2',3-dialdehyde(Compound 8, AVS #2906)

IN VITRO SCREEN [ug/ml]

VIR VR VR+ ID50 CELL MTC TI TI+VSV >0.7 1.3 7.81 L929 <100.00 (12.8 5.9VSV 0.9 1.0 8.M6 L929 100.00 11.3 3.4AD2 0.2 0.7 57.02 HEP2 32.00 0.6 4.8VV 0.7 0.9 11.45 VERO 100.00 8.7 2.6JBg 0.0 0.7 NOT ACT VERC 100.00 0.0 2.0JBg 0.0 0.7 NOT ACT VERO 100.00 0.0 2.0YF 0.8 0.7 24.68 VERO 320.00 13.0 0.2SFS 0.7 0.9 24.82 VERO 100.00 4.0 14.0RVF 335.83 VERO >250.00 0.7 3.7HIV 0.0 2.1 NOT ACT ATHS 10.00 0.0 )320.00

"-12-

S- e . - . .* 0. a- * - .

Table X. Antiviral Activity of N6 ,N6 -Dimethylaminopurine Riboside-2',3'-dialdehyde (Compound 9, AVS #2543)

YN VITRO SCREEN [ug/mlI

SVIR VR VR+ 1D50 CELL MTC TI TI+AD2 >0.1 NOT ACT HEP2 <32.00 0.0VSV 0.1 31.01 L929 32.00 1.0VV 0.0 NOT ACT VERO 32.00 0.0JE 0.0 NOT ACT VERO 32.00 0.0PT 0.3 1.0 59.82 VERO 100.00 1.7 10.8YF 0.0 0.8 NOT ACT VERO 32.00 0.0 1.9SF 0.5 1.1 10.34 VERO 32.00 3.1 :3.8"HIV 0.5 1.4 NOT ACT ATH8 32.00 0.0 >320.0PT 0.7 30.00 M1,2 3.20 0.1 3.5

Table XI. Antiviral Activity Tubetcin-2'3'-dialdehyde(Co."ound 11, AVS #2907)

IN VITRO SCREEN [ug/all

VIR VR VR+ ID50 CELL NTC TI TI+RVF 206.00 VERO >250.00 1.2 >3.7VSV 0.1 1.3 23.92 L929 32.00 1.3 5.9AD2 0.3 0.7 17.77 HEP2 32.00 1.8 4.8VV 0.0 0.9 NOT ACT VERO 100.00 0.0 2.6JBE 0.0 0.7 NOT ACT VERO 100.00 0.0 2.0SFS 0.9 0.9 16.40 VERO 320.00 19.5 14.0Yr 0.3 0.7 199.91 VERO 320.00 1.6 0.2

"-13-

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Legend to Tables II-XIl

IN VIT•O SCIREE - In vitro test results

VIa VirusSymbol Virus

VEE Venezuelan Equine Encephalouyelitis

Yd Yellow Fever

JBZ Japanese Encephalitis Virus

PIC Pichinde

RVF Rift Valley Fever

SFS Sandfly Fever (Silian)

PT Punts Toro Virus

CCH Crimean-Congo Hemorrhagic Fever

VSV Vesicular Stomatitis Virus

AD2 Adenovirus Type 2

VV Vaccinia

FeLV Feline Leukemia Virus

HIV Human Immunodeficiency Virus

Vi Virus rating calculated by the method of Hoffmas and Sidwell

(Appl. Microbiol. 221 795-801, 1971).

VR+ Virus rating for positive control.

IDSO Inhibitory dose 50. Concentration of the drug that causes

a 501 reduction in virus rc.lication.

CELL Cell Line.

MTC Minimum toxic concentration. The lowest concentration

of the test compound that results in a 501 reduction in

rthe percent survival of viable host cells.

TI Therapeutic index. A measure of the antivirel potential

of a compound calculated as MTC/IDSO.

T1+ Therapeutic index of positive controls.

"-14-

//

IN VIVO SCREEN In vivo test results

VIi Virus (see column 2)

MST Hosts HUS - Mouse

VR Virus rating--a mesure of the in oivo antiviral potential

of the drug. Calculated ant

CEO'.TRIC MTD OF EXPERIMENTAL GROUP"GEOMHTRIC MTD OF PLACEBO GROUP

* VR+ VR for postive control.

DOSE Milligrams per kilogram of body veight

"MTC Maximum tolerated dose

RTE Route of drug administrations IP-intraperitoneal

D Schedule of administrations I - Single dose on day I

"-15-

L ~ ~ ~ ~ ~ ~ ~ ~ ~ V" ov Wmm ~ q~Jal~mltmq~~mq.L~tql~Ltq ft • WV tie b a*Oafs0

Experimental Section

IR spectra :ere recorded on a Perkin Elmer 681 Infrared Spectrophotometer.

•H 1 MR r ectra were recorded on an IBM WP 200 SY instrument. Chemicalshift va eas are reported in 6 relative to Me4 Si. UV spectra wererecorded on a Varian Cary 219 Spectrophotometer. Mass spectra wereobtained in the electron impact mode using a direct insertion probeand recorded on a Hewlett Packard 5995 Gas Chromatographi/Mass Spectrometer.Elemental analyses were performed by Gslbraith Laboratories, KnoxviLle.

TN.

4',5'-Unsaturated adenosine-2' ,3'-dialdehyde, 8 (AVS #2906).

Adenosine-2',3'-dialdehyde 2 (3.0 g, 11.3 ol) in 200 m. of waterwas heated under reflux for one hr under a positive pressure of N2 .The crude reaction mixture, containing approx 50% of 8, as indicated

by analytical HPLC (columns 4 m x 30 cm C7 8 coluuln; mýcbile phase:5 vol. acetonitrile in water) was partially purified on a Warars PrepLC 500A system (columns 57 - x 30 cm C1 cartridges mobile phases3 volZ acetonitrile in water; flow rate: 200 Wainin& detectors refractiveindax). Solvents were removed by rotary evaporation under high vacuum

to a omall volume, followed by lyophilization to give 8 in 85% purity.The above procedure was repeated twice. The product of all three reactionswas combined, dissolved in a minimum amount of hot water (approx 175

_L). and further purified or the Prep LC 500A system as described above.Solvents vere removed as described above to give 1.5 X (17% yield)of 95% pure Of as indicated by analytical HPLC.

Physical and Analytical' Data

Melting Points 1966 (dec)

Analysis: For C1 0 HN 5O 3 .1.181H2 0 (268.47)

Calcd FoundC 44.74 44.63H 4.26 3.86N 26.08 25.72

IR Spectrums KBr. Compatible with ctructure. Broad band at 1100cm'1 typical of nucleoside dialdeh~des.

3

RHR (D2 0),

6 9.21 (a, 1 H, H-3'), 8.28 (s,l H, H-8) 8 18 (s 1 H, H1-2), 3.99-5.97(d, I H -I' JHl' 1H-2' - 5.2 Hz), 5.1775.76 (1, 1 X, 1'5 ,HR2 H .3.2" HxJ), 546-5.44 (d, I H, H1-21, 2 H°I' * 5.2 Na), 5.41-5.40(d,1 H. H-5', 4 *.5' H-S' - 3.2 H0).

UV Spectrums •uax (020) 236 nm (c 15,957)

.Mass Spectrum (EI),

4 m/s 247 (Or'), 218 (W" - CHO).

.. . m..-16-

---------.... . • -- ,............ ...... .... ...... _. .... , ..........

Thin-layer Chromatographyt Eastman 13254 cellulose

Eluent Rf Comment

H120 0.68 HomogeneousEtOH-1 M HH4OAc (703) 0.64 Homogeneous

Code No.:, OP-1-143

Prepared byt S. M. Opit:

Tubercidin-2V,3'-dialdshyde, 11 (AVS #29017)..

Prepared by the general method of Dvonch at S12. To a stirredsuspension of 2.96 g (11.12 ao1) of tubercidin in 120 *L of waterwoo added 2.94 g (12.23 mmol) of periodic acid. The reaction mixture

was stirred for one hr at room temp and then applied to a 1.5 x 20.0ca column of Bio-Rad AG I-X8 anion exchange resin (acetate form).The column wasn washed with water. The self-eluate and washings (400 L)were found to be free of iodate and periodate by starch Iodide testpaper, and were lyophilized to give 2.31 S (68% yield) of 11.

_ _ Physical and Analytical Data

Melting Point: >132* (dec)

Analysis. For C111112M404.l.61120

Caled FoundC 45.08 44.95

PPH 5.23 5.31X- 19.12 18.98

IR Spectrum: K~r. Broad band at 1100 cu'l typical of nucleoside dialdehydee.3

NNR 0p201

5 8.17-8.00 (a, 1 H, U1-2). 7.97-5.91 (N, 2 H, H1-7, 11-8), 5.87-4.86(an, 3 H, H-1. H1-2'. H-3'), 4.50-3.36 (a, 3 H. H1-4'. 2 H-5').

UV Spectrum* Amax (1120) 268 nu (C 11,500).

uls264W),282(M+ + H20)

Thin-layer Ch1roisatoltraphy: Eastman 13254 cellulose

Eluent3.

EL ILI "

rEtsared by: H. T. Nemergut

Material.: I

Tubercidin Sigma Chem Co. Lot #106F-7080Periodic acid Sigma Chem Co. Lot #34F-0351Anion exchange resin Bio-Rad AG 1-X8 Control #28817(acetate form)

I'

S... -.- 18- --

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Literature Cited

1. A. J. Grant and L. M. Lerner, J. Med. Chem., 23, 795 (1980).

2. W. Dvonch, H. Fletcher, III, F. J. Gregory, K-N. H. Healy, G. H.Warren, H. Z. Album,. Cancer Res., 26, 2386 (1966).

3. F. Hanuake and F. Cramer. Carbohy r. Res., 54, 75 (1977).

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LiAst of Personnel Supported by the Contract PuJ. P. Neenan. Ph. D. - Principal Invistlgator

Sumittada H. Opitz, Ph.D. - Senior Research Associate

Maureen S. May, Ph.D. - Research Associate

Daniel A. Hendelson - Undergraduate Co-op Student

Linda H. Eckel Undergraduate Co-op Student

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DISTRIBUTION LIST

S copies CommanderUS Army Medical Research Institute of Infe.tious DiseasesATT"s SCRD-UIZ-NFort Detrick, Frederick, ND 21701-5011

I copy CommanderUS Army Medical Research and Development CommadATTNs SCIUI-W-SFort Detrick, Frederick, Maryland 21701-5012

2 copies Defense Technical Information Center (DTIC)AT1T DTIC-DDACCameron StationAlexandria, VA 22304-6145

I copy Dean

School of MedicineUniformed Services University of the Health Services4301 Jones Bridge RoadBethesda. MD 20814-4799

I copy CommandantAcadedy of Health Sciences, US ArmyATTNJ AHS-CDNFort Sam Houston, U' 78234-6100

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