Synthesis of 131I, 125I and 82Br labelled (E)-5-(2-Halovinyl)-2′-deoxyuridines

Int. J. Appl. Radiat. lsot. Vol. 35, No. 11, pp. 1049-1052, 1984 Printed in Great Britain. All fights reserved

0020-708X/84 53.00+0.00 Copyright ~ 1984 Pergamon Press Ltd

Synthesis of 131I, 1251 and 82Br Labelled (E)-5-(2-Halovinyl)-2'-deoxyuridines JOHN

S A M U E L t, E D W A R D

E. K N A U S t*, L E O N A R D LORNE TYRRELL 2

I. W l E B E l and D.

Faculties of ~Pharmacy and "Medicine, University of Alberta, Edmonton, Alberta, Canada T6CJ 2N8 (Received 21 November 1983)

Radiohalogenated (E)-S-(2-iodovinyl)-2'-deoxyuridine (IVDU, 4) and (E)-S-(2-bromovinyl)-2'deoxyuridine (BVDU, 5) were synthesized by reaction of (E)-S-(2-carboxyvinyl)-2"-deoxyuridine (1) with radiolabelled iodide or bromide in the presence of chloramine-T. A "no-carrier-added" synthesis of [mI]IVDU was completed within 30 rain providing a radiochemical yield of 65%. Alternatively, radioactive iodine was incorporated into IVDU using a halogen isotope exchange reaction catalyzed by cuprous ion. [S2Br]BVDU was also prepared by direct neutron activation of unlabelled BVDU.

Introduction Herpes Simplex virus type 1 (HSV-I) is one of the most common causes of fatal sporadic encephalitis in man. (~''~)The successful treatment of HSV encephalitis requires early diagnosis and initiation of chemotherapy using adenine arabinoside (vidarabine) before the patient goes into a coma. °'4) Despite attempts at devising non-invasive diagnostic tests, brain biopsy is still the only definitive means of diagnosing HSV encephalitis. 15-7) A new approach using radiolabelled antiviral drugs as non-invasive probes has recently been proposed. (~) (E)-5-(2-IodovinyD-2'-deoxyuridine (IVDU, 2) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, 3) arc among the most potent agents active against HSV-1.19) Their antiviral activity is attributed to selective phosphorylation by HSV-I encoded thymidine kinase. °°) Therefore, these compounds are expected to selectively localize in HSV-I infected tissue by metabolic trapping. This selective uptake could be used as a basis for the development of a non-invasive diagnostic method using brain imaging techniques. Biological investigations using this approach prompted the development of radiochemical methods applicable to the synthesis of"no-carrier added" and "high specific activity" IVDU and BVDU labelled with short-lived isotopes of iodine and bromine such as t-')I (tl~2, 13.26 h), 13tI (tla, 8.05 d), S2Br (tl,2, 35.34 h), 77Br (tt.,2, 57.04 h), and 75Br (ttrz, 95.5 rain). The method reported for the preparation of ~25I(tt,,., 60.14 d) labelled IVDU (u) does not permit "no carrieradded" synthesis and is not suitable for the incorporation of t23I. We now report a facile procedure for the * Author to w h o m correspondence should be addressed.

synthesis of radiohalogenated IVDU and BVDU using the chloramine-T method. °2-~5) The procedure described is suitable for incorporation of short-lived isotopes such as ~:3I, 77Br, 75Br and is applicable to "no-carrier-added" syntheses. Alternative procedures for the preparation of [13q]IVDU (4) by a halogen isotope exchange method °6) and [S2Br]BVDU (5) by direct neutron activation are also described. Experimental Material and methods

(E)-5-(2-Carboxyvinyl)-2'-deoxyuridine (1), (E)-5(2-iodovinyl)-2'-deoxyuridine (IVDU, 2) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, 3) were synthesized according to the literature procedures °~) and were characterized by m.p., u.v. and IH-NMR spectra. These samples of IVDU and BVDU served as the authentic samples for comparison with those obtained in the radiochemical syntheses. All chemicals used were of reagent grade quality, unless otherwise stated. Solvents were dried using routine methods and fractionally distilled before use. "No-carrier-added" radioactive sodium iodide (t3tI, t25I) was supplied by Edmonton Radiopharmacy Centre, Edmonton, Canada. S-'Br-NH4Br was prepared from enriched NH4Br (97.8% enriched StBr, Oak Ridge National Laboratories, U.S.A.) by the S~Br (n,7) S"Br nuclear reaction at the University of Alberta SLOWPOKE Reactor Facility at a neutron flux of 10~:ncrn-2s -~. A typical yield was 2.3 MBq mg -~ of NH4Br for a 4 h irradiation. Proton magnetic resonance spectra (~H-NMR) were recorded on a Bruker WH-200 spectrometer. Ultraviolet spectra were recorded on a Unicam SP

1049

1050

JOI-L~ SAMUELet al.

800 spectrometer. Thin layer chromatography separations were carded out on Whatman MK 6F microslides (Whatman Inc., New Jersey, U.S.A.) using methanol:chloroform (15:85) as the solvent. Thin layer radiochromatography (TLR.C) was performed on a Berthold LB 2832 Automatic TLC Linear Analyzer equipped with a Berthold LB 281 proportional counter and a Canberra series 40 Multichannel Analyzer. High pressure liquid chromatography (HPLC) separations were performed on a Whatman Partisil-10 CCS/C8 reverse phase M-9 preparative column (9.4ram i.d. x 25crn length) using HPLC grade methanol:water (40:60 by volume) as eluant at a flow rate of 2 mL rain- ~. Ultraviolet absorption (2:298 nm for IVDU and 296 nm for BVDU) and ~-ray detection systems were used for detection of HPLC eluants. Radioactivity measurements were determined with a Picker Dose Calibrator and a Beckman LS 9000 liquid scintillation counter. The radiochemical yields reported are those obtained at the end of synthesis.

Synthesis of [ISlL 125I]-(E)-5-( 2-1odovinyl). 2'.deoxy. uridine (IVDU, 4) (a) Chloramine-T method. (i) High specific activity synthesis: A solution of sodium iodide (50/~g, 0.33 ~mol) in ethanol (100/aL) and potassium acetate (50/~g) in ethanol (50/~L) were added to a 1 mL reaction vial and the solvent was removed under a stream of nitrogen. [~3tI or t2sI]Sodium iodide (118.4 MBq) in ethanol (500/aL) was then added and the volume was reduced to about 20/aL. (E)-5-(2-Carboxyvinyl)-2'-deoxyuridine (ling, 3.3 /~mol) in dry dimethyl formamide (DMF, 100/~L) was added to the reaction vial and the reaction mixture was stirred for 5 rain using a magnetic stirrer. The reaction was initiated by addition of chloramineT (200/~g) in dry DMF (20#L) and stirring was continued at room temperature for 8 h. Radiochromatography (TLRC) analyses indicated that the reaction was complete. The solvent was removed under a stream of nitrogen and the residue purified by HPLC. The radioactive fraction which corresponded to that of an authentic sample with a retention time of 20 rain afforded IVDU (4) (88 #g, 69% chemical yield; 80 MBq, 67.2% radiochemical yield, specific activity 344GBq mmol-~). When the reaction was terminated l h after addition of chloramine-T and the product purified as described above, IVDU was obtained in 58.8 and 57.1% chemical and radiochemieal yield respectively. A cold synthesis of IVDU was carried out using the chloramine-T method to confirm the chemical identity of IVDU. The product was identical (R/, u.v., ~H-NMR) to that of an authentic sample.°7) (ii) "No-carder-added" synthesis: [t~II]IVDU was synthesized as described in (i) without addition of cold sodium iodide using 37 MBq [~3q]sodium iodide. The reaction was complete within 30 min. The prod-

uct was purified by HPLC to give [~I]IVDU (24 MBq, 657,~ radiochemical yield). (b ) Halogen isotope exchange method. A solution of IVDU (70/~g, 0.18 #tool), [t3~I]sodium iodide (3.7 MBq) and cuprous chloride (2 #g, 0.02/~mol) in dry DMF (20/~L) were heated at 70-80°C in a reaction vial. The incorporation of radioactivity into IVDU was monitored by radiochromatography (TLRC). The maximum incorporation of radioactivity into IVDU was achieved after 20 h at which time the solvent was removed. The residue was purified by HPLC as described above to yield IVDU (38/~g, 54.3% chemical recovery; 1.67 MBq, 45.1% radiochemical yield; specific activity 16.7 GBq mmol- ~).

Synthesis of [S:Br]-(E)-5-(2-Bromovinyl)-2"-deoxyuridine (BVDU, 5) (a ) Chloramine- T method. A solution of (E)-5-(2-carboxyvinyl)-2'-deoxyuridine (1, 2 mg, 6.1/~mol), potassium acetate (ling), [~ZBr]ammonium bromide (~300/~g, 3.1/~mol, 0.72MBq) and chloramine-T (2 rag) in dry DMF (200/~L) and ethanol (20/~L) were stirred at room temperature using a magnetic stirrer. The reaction was complete within 10 min as indicated by radiochromatography (TLRC). The solvent was removed and the residue purified by HPLC. The radioactive fraction corresponding to that of an authentic sample with a retention time of 18 min afforded BVDU (5) (727 pg, 72% chemical yield, 0.5 MBq; 69.8~, radiochemical yield; specific activity 227 MBq mmol-t). A cold synthesis of BVDU was carried out using the above method to confirm the identity of BVDU. The product was identical (R:, u.v. ~H-NMR) to that of an authentic sample.(iT) (b ) Direct neutron activation. A sample of BVDU (1 rag) (containing natural abundance bromine) was irradiated in a double plastic vial at a neutron flux of 10J2ncm-Zs -~ for 4h. The sample was allowed to stand for 24 h prior to purification by preparative HPLC as described above. The radioactive fraction having a retention time of 18 min showed identical TLRC, HPLC and u.v. SPectral characteristics to that of the authentic sample of BVDU. The specific activity of the product calculated to the end of irradiation was 32 MBq mmoi -~. Radiolytic decomposition was estimated to be less than 3%. The radioactivity associated with BVDU was 30% of the overall activity produced.

Results and Disctmsion Unlabclled IVDU 2 and BVDU 3 were synthesized by the stereospecific reaction of I with the corresponding N-halosuccinimide (routes A and B, Fig. I).(~) This reaction is not suitable for radiohalogenation since it would require synthesis of the appropriate radiohalogenated N-halosuccinimide. It was therefore of interest to develop alternative meth-

Radiohalogenated halovinyl deoxyuridincs

O

0 A Hx / I HN~ "-,y~C=Cx

[13t I , l i S I ]

ONJ

[131I ] - NaI./CuCl 70 -80"

®

4

I

OH

®/

[ 131I, 125Z] NaZi

O

Chloramine-T

~j HN/

[81Br ]

I051

/

OH N--Todosuccinimide

H /CO2 H t~'C=C~ H

NH4Br

[

n,Y

[82 8rl-NH4 B r /

Chloramine-

~

I

OH

T

- Brornosucc inimide

O

[82 B r ]

HN

-

\H

U

O,~~ N

®

Br

H" _ /

c--~ H

O n,l~

OH Fig. I. Synthesis of radiohalogenated IVDU (4) and BVDU (5).

ods which included (i) radioiodination and radiobromination using chloramine-T (routes C and D); (ii) cuprous ion catalyzed halogen isotope exchange for radioiodination (route E); and (iii) direct neutron activation of cold BVDU (route F). The radiochemical yield of 4 prepared using a variety of reaction conditions is summarized in Table 1. The reaction time could be reduced to 1 h without a substantial reduction in radiochemical yieM for the "carrier added" syntheses using chloramine-T. The "no-carrier-added" reaction was complete within 30 min affording 4 in 65~ radiochemical yield. This short reaction time makes it a useful synthetic method for the ~yntheses of [mI]IVDU (t~. for

tuI = 13.26 h) for imaging studies using single photon emission computed tomography (SPECT). The cuprous ion catalyzed exchange reaction (route E) required heating at 70-80°C for a prolonged time (20 h). The low chemical recovery and radiochemical yield may be due to thermal decomposition. The stereospecificity of the radioiodination reaction observed is consistent with previous studies involving halogen isotope exchange reactions for other vinyl iodides. (tt) The exchange reaction is believed to proceed via generation of a small quantity of Cu~3~I, which undergoes ligand exchange with 2.(~ There is less radiation hazard since this method does not generate a volatile radioiodine species. This exchange

1052

JOHN SAMUEL et al. Table 1. Radiochemical synthesis of radioiodinated IVDU

Method Chloramine-T Chloramine-T Chloramine-T Halogen isotope exchange

Specific activity

Temperature (°C)

344 GBq mmol -t 344 GBq retool- 1 No carrier added 16.7 GBq mmol-

25

Chemical yield,

Time

R,adiochemical yield (~)

recovery C'o)

8h

67.2

69.0

25

Ih

57.1

58.8

25

30 rain

65.0

--

70-80

20 h

45.1

54.3

reaction is limited to the preparation of low and medium specific activity products. SZBr was used as a model isotope of radiobromine in these studies since it is easily prepared at the University of Alberta SLOWPOKE reactor Facility. The neutron activation of [SIBr]NH4Br using the ~lBr (n, y)82Br nuclear reaction at a flux of 101' n cm -2 s -I for 4 h gave [S2Br]NH4Br with a specific activity of 2.3MBqmg -~. It would be possible to increase the specific activity to 6 M B q m g -~ with a subsequent 16h irradiation at a neutron flux of 0.5 x 10t'-ncm-2s -~. The reaction of 1 with [SZBr]NH4Br in the presence of chloramine-T gave [SZBr]BVDU (5) (69.8% radiochemical yield; specific activity 227 MBq mmol-I). The short reaction time ( < 1 0 m i n ) makes this a suitable method for the incorporation of the positron emitter 75Br (t~n, 95.5 min). The direct neutron activation was carried out using BVDU which contained natural abundance bromine. A 24 h cool-off time was required to allow decay of s°'~Br (tj :, 4.4 h) before the irradiated sample was purified. Although radiolytic decomposition was minimal (<3%), only 30% of the overall activity produced was associated with [S2Br]BVDU. This is due to the Szilard-Chalmers cleavage reaction which frequently occurs during neutron activation of organic halides. (~s~The specific activity of [8~Br]BVDU calculated to the end of irradiation was very low (32 MBqmmol-t). The direct irradiation method is not the method of choice for the synthesis of [82Br]BVDU, since most in vivo studies require a much higher specific activity product. The radiohalogenated halovinyl deoxyuridines prepared are being evaluated as potential radiopharmaceuticals for the non-invasive diagnosis of herpes encephalitis. The preliminary in vitro uptake studies showed selective localization of IVDU in HSV-I infected cells. (~9~Other studies which include quantitative tissue distribution studies and brain imaging using animal models are in progress. Acknowledgements--We are grateful to the Medical Research Council of Canada (Grant MA-5965) for financial support of this research and the Alberta Heritage Founda-

tion for Medical Research for a Studentship to one of us (J.S.). We thank the Alberta Mental Health Research Fund for a grant to one of us (D.L.T.). We also thank Dr R. J. Flanagan for helpful suggestions concerning the cuprous ion catalyzed exchange reaction and Mr P. Ford for performing the bromine irradiations.

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