Reactivity and Selectivity of F - American Chemical Society

Chapter 10

Reactivity and Selectivity of F Toward 3,4,6-Tri-O-acetyl-D-Glucal in Anhydrous HF: Synthesis of[18F]2-Fluoro-2-deoxy-β-D-allose and Its Use for the Detection of a Breast Tumor Downloaded by PURDUE UNIV on November 25, 2016 | http://pubs.acs.org Publication Date: January 1, 2009 | doi: 10.1021/bk-2009-1003.ch010

2

1

1,2,

2,

Rezwan Ashique , Raman Chirakal *, Gary J. Schrobilgen *, Donald W. Hughes , Troy Farncombe , Karen Gulenchyn , Renée Labiris , Tracey Truman , and Chantal Saab 2

1

3

1

3

4

1

Department of Nuclear Medicine, Hamilton Health Sciences, Hamilton, Ontario L 8 N 3Z5, Canada Department of Chemistry, McMaster University, Hamilton, Ontario L8S 4 M 1 , Canada Department of Medicine, McMaster University, Hamilton, Ontario L8S 4L8, Canada M c M a s t e r Centre for Pre-clinical and Translational Imaging, Hamilton, Ontario L 8 N 3Z5, Canada 2

3

4

Because of growing interest in the biological properties of D-allose, its F-labeled analogue, [ F]2-fluoro-2-deoxy-β-D -allose ([ F]2-FDβA), was synthesized to probe its biological activity. A fast and efficient two-step, regio-, and stereoselective synthesis of [ F]2-FDβA was achieved by electrophilic fluorination of 3,4,6-tri-O-acetyl-D-glucal in anhydrous HF using [ F]F and [ F]CH COOF. The fluorocarbohydrate was characterized by multi-NMR spectroscopy and mass spectrometry. Small animal PET imaging was used to study the in vivo behavior of [ F]2-FDβA. Preliminary studies using Polynoma Middle Τ mice showed that [ F]2-FDβA is a promising radiotracer for the detection of breast tumors. 18

18

18

18

18

18

2

3

18

18

© 2009 American Chemical Society

Gakh and Kirk; Fluorinated Heterocycles ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

211

212

Downloaded by PURDUE UNIV on November 25, 2016 | http://pubs.acs.org Publication Date: January 1, 2009 | doi: 10.1021/bk-2009-1003.ch010

Introduction Rare sugars are defined by the International Society of Rare Sugars (ISRS) as monosaccharides that are infrequently encountered in nature. Because of their rarity and their laborious and expensive chemical syntheses, the biological properties of rare sugars have not been investigated in detail. Recently, an effective strategy for the large-scale production of these sugars has been developed (1,2) and interest in studying their properties is rapidly growing. Recent studies on the biological properties of one such rare sugar, D-allose (Figure 1), the C3 epimer of D-glucose, show that it provides very effective protection against neutrophil-related postischemic injury of liver tissue (3), inhibits segmented neutrophil production (4), and lowers platelet count without detrimental side effects (4). Such characteristics suggest that D-allose may be a potential therapeutic agent against cancer. Significant advances in our understanding of the biochemical behavior of D-allose can be made using its F-labeled analogue, namely [ F]2-fluoro-2-deoxy-D-allose, in conjunction with Positron Emission Tomography (PET). Deoxyfluorinated analogues allow studies of the transport and metabolism of the corresponding parent sugars and aid in isolating specific biochemical reaction sequences from general metabolic pathways, especially in the functional imaging sciences (5). For example, the F-labeled analogue of D-glucose, [ F]2-fluoro-2-deoxy-D-glucose ([ F]2FDG), is a well-established tracer in PET that is used to measure the glucose utilization rates in normal tissue and tumors. By analogy with D-glucose and [ F]2-FDG, it is anticipated that [ F]2-fluoro-2-deoxy-D-allose should be an excellent tracer for the study of the in vivo behavior of D-allose (Figure 1). The first synthesis of 2-fluoro-2-deoxy-D-allose was reported by Johansson and Lindberg (Scheme 1) ( 3.1

% , F , 8.9

}

Jm,m, 8.2

H

2

3

Jm.H4, 3.0

V ,H5, 10.0

4

J\I*,F, 16

H4

3

-^H5,H6» 2.3

3

·Λ|5,Η6'> 5.8

2

^H6.H6S 12.3

"The abbreviations denote doublet of doublets (dd) and doublet of doublets of doublets (ddd). *This multiplet appears as a doublet of "triplets" because J 3,H4 « JH2,H3Reproduced with permission from ref 14. Copyright 2006 Elsevier. 3

3

H

Table II. l3

1 3

C N M R Parameters for 2 Π ) β Α

C chemical shift" (ppm)

multiplicity''

Coupling constant (Hz)

CI

91.93

(96.4)

d

V ,.F, 24.5

C2

90.67

(74.2)

d

'JC2,F, 185.5

C3

70.09

(74.3)

d

C4

67.05

(69.5)

d

C5

74.47

(76.5)

s

C6

61.75

(64.1)

s

C

2

-/ 3,F, 15.9 C

3

^C4,F> 5.5

°Chemical shifts in parentheses are those of β-D-allose (2/). The abbreviations denote doublet (d) and singlet (s). Reproduced with permission from ref 14. Copyright 2006 Elsevier. A


224 Mass Spectrometric Characterization Negative ion electrospray ionization (ESI) mass spectrometry of the reaction products resulting from Scheme 4 corroborated the N M R findings showing that 2-FDPA was the major product. The ESI mass spectrum showed the deprotonated molecular ion, [M-H]~ at m/z 181. However, the base peak in the spectrum appeared at m/z 217, which corresponds to the chloride adduct of neutral 2-FDPA. It is noteworthy that 2-FDpA gave a fragmentation pattern that was very similar to the combined fragmentation patterns of 2-FDG and 2-FDM formed by the fluorination of T A G in CFC1 . Downloaded by PURDUE UNIV on November 25, 2016 | http://pubs.acs.org Publication Date: January 1, 2009 | doi: 10.1021/bk-2009-1003.ch010

3

l8

Chromatographic Properties of l F]2-FDpA It is imperative to establish the identity, chemical purity, and radiochemical purity of any tracer used for in vivo and in vitro studies. The identity of [ F]2-FDpA was established by H , C , and F N M R spectroscopy and mass spectrometry, after samples were allowed to decay for a minimum of 48 h. For routine use, only radio-HPLC (stationary phase: Waters Carbohydrate Analysis Column, 3.9 χ 300 mm, 10 μ; mobile phase: a 95% C H C N and 5% H 0 solvent mixture at 2.0 mL/min) and radio-TLC (stationary phase: Alltech silica gel 60 T L C aluminium sheet, 200 μ; mobile phase: a 95% C H C N and 5% H 0 solvent mixture) were used to determine the identity and radiochemical purity of [ F]2-FDpA. As expected from their structural similarities, [ F]2-FDPA had chromatographic properties very similar to those of [ F]2-FDG and [ F]2-FDM. Figures 6 and 7 show the radio-HPLC and radio-TLC chromatograms, respectively, of [ F]2-FDpA and a mixture of [ F]2-FDG and [ F]2-FDM. The retention time (t ) of [ F]2-FDpA was 3.83 min (Figure 5a), whereas those of [ F]2-FDG and [ F]2-FDM were 3.75 min (Figure 5b). The dark spots on the T L C plates (Figure 6) correspond to the F radioactivity, which gave rise to the peaks on the T L C chromatograms. The R value of [ F]2-FDPA, 0.37, was also very close to those of [ F]2-FDG and [ F]2-FDM (0.36). The radiochemical purity of [ F]2-FDpA was 96 ±3 and 85 ±7%, as determined by radio-HPLC and radio-TLC, respectively. 18

!

, 3

19

,8

3

2

3

2

18

18

18

,8

18

,8

!8

R

,8

,8

18

r

18

18

,8

18

Proposed Mechanism for the Synthesis of 2-FDPA Using F2 Fluorinations of T A G and D-glucal by F in polar solvents and aqueous solutions usually result in difluorinated intermediates in which F is added across the double bond to give a ^«-arrangement (8,9,22). The absence of an antiarrangement is attributed to the inability of fluorine, in contrast with other halogens, to form a halonium ion. Therefore, the initial fluorocarbonium ion intermediate, formed by electrophilic attack of F at C2, is not bridged (23). The 2

2

2


225


tensiity (mV)

450 -

c

[ F]2-FDpA 18

375 300 225 150 -

LL

75 -

00

3

4

5

6

7

8

10

Retention Time (min)

600 [ F]2-FDG and [ F]2-FDM 18

525 Ε* m ΒC

jC LL

CO

18

450 375 300 225 150 75 0 3

4

5

6

7

8

10

Retention Time (min) Figure 5. Radio-HPLC chromatograms of (a) the products resulting from the electrophilic fluorination of TAG in aHF and (b) a mixture of[ F]2-FDG and [ F]2-FDM resulting from the electrophilic fluorination of TAG in CFCl . 18

I8

3


226

1


[ T]2-FDpA

50

100

150

200

Distance (mm)

solvent front

origin

[ F]2-FDG and [ F]2-FDM 18

2.0

18

1.5

χ f?

1.0

Ο Ο

0.5 0.0 50

100

150

200

Distance (mm)

solvent front

origin

Figure 6. Radio-TLC chromatograms and plates of (a) the products resulting from the electrophilic fluorination of TAG in aHF and (b) a mixture of [ F]2-FDG and [ F]2-FDM resulting from the electrophilic fluorination ofTAGinCFCh 18

1H


227 fluorocarbonium ion rapidly combines with the F~ counter ion to form the difluorinated intermediate in which the fluorine ligand on CI is susceptible to hydrolysis in dilute acid media. Lundt and Pedersen (12) proposed that the reaction of T A G with aHF (Scheme 3) proceeded by protonation of the Oacetyl oxygen atom bonded to C3. Subsequent cleavage of the Oacetyl group to form acetic acid was likely assisted by attack from the acyloxy group at C4. These workers showed, from a freshly prepared aHF solution of T A G , that the double bond of the corresponding 1,2unsaturated 3,4-dioxolenium ion was initially intact at -70 °C. The H N M R spectrum obtained after a period of 24 h showed that HF had added across the double bond of the dioxolenium ion in a Markovnikov fashion. The introduction of a small amount of water into the reaction medium resulted in hydrolysis of the dioxolenium ion and subsequent epimerization at C3. Fluorinations of T A G in the present study were routinely carried out at -60 °C within 30 min following addition of aHF, thus avoiding significant HF addition to the double bond of the 1,2-unsaturated dioxolenium ion prior to fluorination. It is expected that the formation of 2-FDpA takes place through a difluorinated intermediate, 3,6-di-Oacetyl-2-deoxy-2-fluoro-ot-D-allopyranosyl fluoride (Scheme 5; I), similar to that formed during the reaction of T A G with F in CFC1 (7,8,24). Epimerization at C3 takes place in aHF followed by the addition of F across the double bond in T A G and subsequent removal of HF, resulting in I. The hydrolysis of I then yields 2-FDp A. Because of the predominance of 2-FDpA in the final product solution, it was anticipated that I would be the major reaction intermediate, and would thus give rise to the most intense signals in F N M R spectrum. The F - F C O S Y spectrum of the reaction mixture recorded immediately following fluorination (Figure 7), showed strong cross-peaks between the resonances at -147.8 and -205.2 ppm. These resonances were assigned to the fluorine ligands of the difluorinated intermediate (I) because their chemical shifts were very similar to those observed for the intermediate resulting from the electrophilic fluorination of T A G in CFCI3 (-151.3 and -205.1 ppm) (24).


!

2

3

2

19

,9

19

18

Biological Studies Using [ F]2-FDpA A l l animal experiments were conducted in accordance with the guidelines of the Institutional Review Board, McMaster University. For studies involving biodistribution and the time course of [ F]2-FDPA, male P M T mice were bred with commercial F V B (inbred strain) control females. The mice developed tumors at approximately 5 weeks of age. The results obtained using [ F]2-FDpA were compared with those using [ F]2-FDG. 18

l8

t8



δ"

δ

Scheme 5, A proposed partial reaction mechanism for the synthesis of 2-ΕΟβΑ by electrophilic fluorination of TAG in aHF. (Reproduced with permission from ref 14. Copyright 2006 Elsevier.)


+

00


229

Figure 7. The F- F gradient COSY spectrum (CD CN solvent at 25 X!) of the intermediates resulting from the electrophilic fluorination of TAG in aHF. 3


230 The mice were injected with 10-18 mBq (0.3-0.5 mCi) of either [ F]2~FDpA or [ F]2-FDG in about 0.2 mL of saline solution through the tail vein. Animals were sedated and maintained with 1-2% isofluorane and whole body dynamic PET scans were acquired from 5 min before to 120 min after injection using a Philips Mosaic small animal PET scanner. Biodistribution studies were carried out by euthanizing the mice by cardiac puncture using 0.03 mL of Euthanol followed by dissection and the removal of the tissue samples. Tissue samples were weighed and their F content measured using a Packard Cobra II Auto-Gamma Counter. The tissue uptake indices were calculated by dividing activity per gram of tissue by injected activity per body weight. Tissue : blood ratios were calculated by dividing the tissue uptake index by the blood uptake index. The whole body PET image of an F V B control mouse 45 min after injection of [ F]2-FDpA (Figure 8) showed significant uptake in the kidney and bladder and no uptake in the brain. The time course of F in a control mouse after administering [ F]2-FDPA showed that almost all of the F activity was present in the bladder within 60 min after injection (Figure 9). Moreover, F uptake in the bone was not observed even after 120 min, which proved that [ F]2-FDpA was not catabolised in vivo to free F~. ,8

,8


18

!8

, 8

l8

, 8

18

,8

,8

Because of the very similar structures of D-glucose and D-allose, one would expect very similar biological properties for both tracers. A comparison of the biodistribution of [ F]2-FDpA with that of [ F]2-FDG in control mice, sacrificed at different times (30-90 min; Table III), however, showed the in vivo properties of the two tracers to be markedly different, that is, the F organ distribution data (Table III) showed significant differences in the uptake in the heart, brain, and bone. The standard errors in the uptake indices (values in parentheses) are rather large because they are average values obtained from different experiments in which the animals were sacrificed at different times. The time course of F in a tumor-bearing P M T mouse after injection of [ F]2-FDpA is shown in Figure 10. The tumor can be clearly visualized within 30 min after tracer injection. Biodistribution studies showed a high level of [ F]2-FDPA uptake in the tumor when compared with the non-targeted tissues (Table IV). The tumor : blood ratio was 10 ±6; the high standard error results from the differences in sacrifice time (80 ±20 min). Figure 11 compares the F uptake between [ F]2-FDpA and [ F]2-FDG using a tumor-bearing P M T mouse, which was injected with each tracer on two consecutive days. Although uptakes of both tracers were clearly visible in the tumor, unlike that of [ F]2-FDG, accumulation of [ F]2-FDpA was not visible in the non-targeted tissues, except the bladder. Hence, [ F]2-FDPA has the potential to provide a better signal-to-noise ratio for tumors. 18

,8

, 8

, 8

18

I8

, 8

,8

18

18

,8

18



231

Figure 8. Whole body PET image of a FVB control mouse (coronal view on the left and sagital view on the right) 60 min after injection of [ F]2~FDfiA. !8

Figure 9. Time course of F in a FVB control mouse after injection of[ F]2-FDpA. 18


232

18

18

Table III. Biodistribution of [ F | 2 - F D p A vs. [ F]2-FDG in Control Mice" ,8

l8

[ F]2-FDPA


Tissue

[ F]2-FDG

Uptake Index

Tissue : Blood

Uptake Index

Tissue : Blood

Lung

0.16(0.03)

2.7(1.1)

0.20 (0.03)

6.6 (0.4)

Liver

0.16(0.07)

2.1 (0.5)

0.08 (0.01)

2.48 (0.09)

0.3 (0.2)

2.0 (0.2)

0.20 (0.05)

6.2(1.1)

Heart

0.23 (0.01)

4.0(1.8)

2.5 (0.6)

81 (13)

Brain

0.041 (0.001)

0.8 (0.3)

0.34 (0.04)

11(2)

Bone

0.08 (0.04)

1.0(0.2)

0.4 (0.1)

14(4)

Blood

0.13(0.08)

1.0 (0.0)

0.031 (0.005)

1.0 (0.0)

Kidney

"Average values obtained using a sample population of three. Standard errors in the values are shown in parentheses.

Figure 10. Time course of F in a tumor-bearing PMT mouse after it was injected with [ F]2-FDfiA. l8


233

,8


Table IV. Biodistribution of [ F]2-FDpA in Tumor-bearing PMT Mice" Tissue

Uptake Index

Tissue : Blood

Lung

0.18(0.04)

3.3 (0.7)

Liver

0.12(0.02)

2.5 (0.6)

Kidney

0.16(0.04)

3.3 (1.007)

Heart

0.30 (0.06)

8.2 (3.4)

Brain

0.045 (0.009)

0.8 (0.2)

Bone

0.10(0.02)

1.9 (0.4)

Blood

0.06 (0.02)

1.0 (0.0)

Tumor

0.3 (0.1)

10(6)

"Average values obtained using a sample population of six. Standard errors in the values are shown in parentheses.

Figure 11. Whole body PET image of a tumor-bearing 18

PMT mouse that was !8

first injected with [ F]2-FDpA (left) and then with [ F]2-FDG two consecutive days.

(right) on


234 Conclusion The present study has employed F and CH COOF to develop reliable and rapid two-step highly regio- and stereoselective syntheses of 2-FDpA by electrophilic fluorination of TAG in aHF. The salient features of these reactions are: (1) high regioselectivity, (2) high stereoselectivity, which is unique for electrophilic fluorination, (3) in situ epimerization at C3, (4) high RCY (in the case of [ F] ) for electrophilic fluorinations, (5) short syntheses times, which are ideal for F-labeling, and (6) no formation of [ F]2-FDG or [ F]2-FDM. It is noteworthy that the chromatographic properties of [ F]2-FDPA are almost identical to those of [ F]2-FDG and [ F]2-FDM, both of which, however, show markedly different in vivo behaviors. Therefore, high regio- and stereoselectivity of the reaction is essential and underscores one of the advantages of using aHF as the reaction medium. Preliminary in vivo studies using FVB control and tumor-bearing PMT mice showed significant differences between the uptakes of [ F]2-FDpA and [ F]2-FDG. Whole body PET images of PMT mice showed retention of [ F]2-FDpA in the tumor with corresponding high tumor to blood ratios, indicating that [ F]2-FDpA is a promising tracer for the detection of breast tumors. 2

3

,8

2

,8

!8

,8

18


18

18

18

,8

I8

,8

Acknowledgement We thank Ms. Carol Dunne, Manager of Nuclear Medicine and Hamilton Health Sciences for financial support and for providing hospital facilities for this research. We also thank Dr. Hélène Mercier for her help with the preparation of the manuscript.

References 1.

2. 3. 4. 5.

Morimoto, K.; Park, C.-S.; Ozaki, M.; Takeshita, K.; Shimonishi, T.; Granström, T.B.; Takata, G.; Tokuda, M.; Izumori, K. Enzyme Microb. Technol. 2006, 38, 855-859. Takeshita, K.; Suga, Α.; Takada, G.; Izumori, K. J. Biosci. Bioeng. 2000, 90, 453-455. Hossain, M.Α.; Izuishi, K; Tokuda, M.; Izumori, K; Maeta, H. J. Hepatobiliary Ρancreat. Surg. 2004, 11, 181-189. Arnold, E.C.; Silady, P.J. U.S. Patent 5,620,960, 1997. Buethium-Baumann, B.; Hamacher, K.; Oberdorfer, F.; Steinbach, J. Carbohydr. Res. 2000, 327, 107-118.


235 6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Johansson, I.; Lindberg, Β. Carbohydr. Res. 1966, 1, 467-473. Ido, T.; Wan, C.-N.; Casella, V.; Fowler, J.S.; Wolf, A.P. J. Label. Comp. Radiopharm. 1978, 14, 175-183. Ido, T.; Wan, C.-N.; Fowler, J.S., Wolf, A.P. J. Org. Chem. 1977, 42, 23412342 Adam, M.J. J. Chem. Soc., Chem. Commun. 1982, 730-731. Ishiwata, K; Ido, T.; Nakanishi, H.; Iwata, R. Appl. Radiat. Isot. 1987, 38, 463-466. Lundt, I.; Pedersen, C. Acta Chem. Scand. 1966, 20, 1369-1375. Lundt, I.; Pedersen, C. Acta Chem. Scand. 1970, 24, 240-246. Lundt, I.; Pedersen, C. Acta Chem. Scand. 1971, 25, 2749-2756. Ashique, R.; Chirakal, R.V.; Hughes, D.W.; Schrobilgen, G.J. Carbohydr. Res. 2006, 341, 457-466. Chirakal, R.; Vasdev N.; Schrobilgen, G.J.; Nahmias, C. J. Fluorine Chem. 1999, 99, 87-94. Vasdev, N.; Chirakal, R.; Schrobilgen, G.J.; Nahmias, C. J. Fluorine Chem. 2001, 111. 17-25. Van Rijn, C.J.S.; Herscheid, J.D.M.; Visser, G.W.M.; Hoekstra, A. Int. J. Appl. Radiat. Isot. 1985, 36, 111-115. Shiue, C.-Y.; Salvadori, P.A.; Wolf, A.P.; Fowler, J.S.; MacGregor, R.R. J. Nucl. Med. 1982, 23, 899-903. Rozen, S.; Lerman, O.; Kol, M. J. Chem. Soc., Chem. Commun. 1981, 443444. Ehrenkaufer, R.W.; Potocki, J.F.; Jewett, D.M. J. Nucl, Med. 1984, 25, 333337. Sigma-Aldrich product information webpage for β-D-allose; http://www.sigmaaldrich.com/spectra/fnmr/FNMR005499.PDF Adam, M.J.; Pate, B.D.; Nesser, J.-R.; Hall, L.D. Carbohydr. Res. 1983, 124, 215-224. Pengles, A.A. Adv. Carbohydr. Chem. Biochem. 1981, 38, 195-285. Diksic, M.; Jolly, D. Carbohydr. Res. 1986, 153, 17-24.


Reactivity and Selectivity of F - American Chemical Society

Recommend Documents