The synthesis of a 5-HETE photoa�nity ligand

Goutam Saha,a Subhash P. Khanapure,a,y William S. Powellb and Joshua Rokacha,*aClaude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 W. University Blvd.,

Melbourne, FL 32901, USAbMeakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St.,

Montreal, QC H2X 2P2, Canada

Received 30 May 2000; revised 14 June 2000; accepted 16 June 2000

Abstract

The design and synthesis of a photoa�nity ligand 26, targeted at the 5h-dh, was accomplished. Thesynthesis was e�ected by two new synthetic routes which focus on the o-hydroxy 5-HETE derivative 12 asa pivotal synthon. Preliminary results show ligand 26 to be an excellent substrate for the 5h-dh. # 2000Elsevier Science Ltd. All rights reserved.

Keywords: 5-HETE; 5-oxo-ETE; o-functionalized-5-HETE; 5h-dh.

5-Oxo-ETE 3, a novel metabolite of arachidonic acid (AA) 1, is a potent chemotactic agent forhuman eosinophils which, unlike neutrophils, responds only weakly to LTB4.

1ÿ3 We have shownthat 5-oxo-ETE induces pulmonary in®ltration of eosinophils in vivo, raising the possibility thatit may play an important role in the recruitment of these cells to the lungs in asthmatics.4 Also,there is substantial evidence that the e�ects of 5-oxo-ETE on granulocytes are mediated by aninteraction with a speci®c G-protein-linked receptor.5ÿ8

Scheme 1 shows the enzymatic machinery associated with the 5-oxo-ETE biosynthesis,metabolism and receptor interaction leading to biological activity. 5-Oxo-ETE 3 is synthesized by

0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PI I : S0040-4039(00 )01064-9

Tetrahedron Letters 41 (2000) 6313±6317

Scheme 1. Four proteins involved in the biosyntheses, metabolism and biological activity of 5-oxo-ETE

* Corresponding author. Tel: 321 674 7329; fax: 321 952 1818. E-mail: jrokach@®t.eduy Present address: NitroMed, Inc., 12 Oak Park Drive, Bedford, MA 01730, USA.

5-hydroxyeicosanoid dehydrogenase (5h-dh), which is highly speci®c for eicosanoids with ahydroxyl group in the 5-position and in the S-con®guration followed by a 6,7-trans-doublebond.9 Because of the latter requirement, LTB4 is not metabolized by 5h-dh, whereas 6-trans-isomers of LTB4 are relatively good substrates. 5-Oxo-ETE can be metabolized by a �6-reductasein neutrophils to 6,7-dihydro-5-oxo-ETE 4.10

Our ®rst objective in this program is the characterization and puri®cation of 5h-dh. Toaccomplish this we have designed and synthesized a photoa�nity label for this enzyme. We arealso reporting preliminary data indicating that this synthetic ligand is a good substrate for 5h-dh.Design of the photoa�nity label. We have considered two designs, A and B (Scheme 2), for

potential radiophotoa�nity labels for 5h-dh. Design A, which is the subject of this report, hasheteroatoms at C-20. These are less lipophilic than the natural substrate 5-HETE and may ormay not interfere in a detrimental fashion with the binding at the catalytic site of the enzyme. Ifthis were the case, design B is an alternative all-carbon analog of design A, which is lipophilicaround C-20.

We selected as our ®rst option the synthesis of 26.11 Three points are worth commenting uponat this stage: (1) The selection of a phenyl group at the o-end of 5-HETE is predicated byprecedents in the literature in the eicosanoid ®eld.12,13 Although a simpler approach, modi®cationof the free carboxylic acid of 5-HETE is unlikely to result in a ligand with high a�nity for 5h-dh,as preliminary studies have shown that 5-HETE methyl ester is a very poor substrate for theenzyme; (2) compound 26 has a 14,15-saturated double bond, in contrast to 5-HETE 2. Therationale for this is as follows: We are assuming that the 14,15-double bond in the naturalsubstrate 5-HETE occupies a primary binding site with a speci®c location in the enzymatic cavity.By saturating this 14,15-position, we thought that this would allow a looser binding (lessenergetic) at that site and permit the phenyl group to assume its most comfortable conformationand maximum binding energy in the catalytic site; and (3) the third point is the selection of anamide as the linking group to the aromatic portion. This was a risky proposition in that theamide linkage is much more polar than the Me group at the C-20 of 5-HETE. An alternative is tocouple 19 with the aromatic group 23, which would yield 5 and a less polar ester function aroundC-20. For this reason we designed the syntheses shown in Schemes 3 and 4 with 19 as a pivotalintermediate to also allow us to prepare the a�nity label 5.Two new synthetic approaches to 26. In order to implement design 6, we developed two new

synthetic approaches for the synthesis of an o-hydroxy and an o-amino derivative (Schemes 3and 4).The synthesis described in Scheme 3 starts with d-arabinose. Aldehyde 10 was prepared in nine

steps in a much improved synthesis described by us very recently.14 The transformation of 10 to12 proceeded in acceptable yield. The Wittig reaction of 12 and 13 to a�ord 14 is a bit tricky. The

Scheme 2.

6314

excellent yield obtained and reported here requires 3 equivalents of 13. The yields drop to 68%with 2 equivalents and to 38% with 1.2 equivalents. In the last two cases some starting aldehydeis recovered. This may indicate that the initial coupling with the aldehyde is slow. This situationprobably allows secondary reactions of the aldehyde to occur under the basic conditions,explaining the lower yield. a,b-Unsaturated aldehydes are less reactive than saturated aldehydes.

Scheme 3. Reagents and conditions: (a) benzene, re¯ux, 4 h, 78%; (b) LiHMDS, THF, HMPA, ^78�C to rt, 97%;

(c) PPTS, EtOH, rt, 3.5 h, 86%; (d) Dess±Martin periodinane, CH2Cl2, rt, 2 h 82%; (e) LiHMDS, THF, HMPA,^78�C to rt, 75%; (f) PPTS, EtOH, rt, 3.5 h, 78%; (g) PPh3, I2, imidazole, CH2Cl2, 0

�C to rt, 88%; (h) NaN3, acetone,65�C, 16 h, 89%; (i) PPh3, H2O, THF, rt, 14 h, 88%; (j) EDCI, HOBT, NaHCO3, DMF, 0�C to rt, 48 h, 89%; (k) 1N

NaOH(aq.), MeOH, rt, 30 h, then 1N HCl, 93%; (l) TBAF:HOAc (1:1), THF, rt, 50 h, 90%

Scheme 4. Reagents and conditions: (a) dihydropyran, PTSA, 0�C to rt, 2 h, 89%; (b) PPh3, CH3CN, 70�C, 38 h, 94%;(c) LiHMDS, HMPA, THF, ^78�C to rt, 78%; (d) TBAF, rt, 28 h, 94%; (e) PPh3, I2, imidazole, CH2Cl2, 0

�C to rt, 2 h,

92%; (f) PPh3, CH3CN, 70�C, 48 h, 95%; (g) LiHMDS, HMPA, THF, ^78�C to rt, 85%; (h) Me2AlCl, CH2Cl2, ^30�Cto rt, 1.5 h, 75%

6315

The novel aldehyde 16 was prepared in two steps from 14 in good yield. The transformation of14 to 18 is better performed the same day, as 15 and 16 both deteriorate on standing. The cisdouble bond in alcohol 15 seems to isomerize to trans as judged by 1H NMR. However, the cis-and trans-isomers are di�cult to separate. It is not clear what causes this isomerization, which isabout 60% complete upon standing overnight. Aldehyde 16 decomposes on standing. It is ourexperience that b,g-unsaturated aldehydes do not store well, in part due to conjugations of thedouble bond(s) with the carbonyl functions. Transformation of 19 to 20, 21 and then 22 proceedwell. The coupling of 22 and 23 proceeds in high yield. EDCI under the conditions described inthe legend works much better than coupling of DCC in acetonitrile.The ®rst synthesis described in Scheme 3 is linear and approached in an anticlockwise manner

in which C-1 is made ®rst and C-20 last. Its advantage lies in the possibility of using aldehyde 16for the introduction of di�erent o-substituted derivatives.The second synthesis described in Scheme 4 is designed to be more convergent and hence more

e�cient. The syntheses of the two synthons 34 and 12 are approximately of equal complexity.Enzymatic studies. Substitution of 125I for the iodine in 26 will require substantial additional

e�ort. Hence it was essential to know whether 26 is recognized well enough by the 5h-dh to justifysuch e�orts. We have performed the following preliminary studies and, as can be seen, 26 hasturned out to be an excellent substrate for the 5h-dh which bodes well for the labeling studies.Azido-5-HETE 3 (2 mM) was incubated for 60 min with a microsomal fraction from human

neutrophils in the presence of NADP+ and the reaction products were analyzed by reverse-phase-HPLC using a Waters Novapak C18 column (3.9�150 mm). The mobile phase consisted of agradient between water:acetonitrile:acetic acid (45:55:0.02) and water:acetonitrile:acetic acid(35:65:0.02) over 30 min at a ¯ow rate of 1 ml/min. As shown in Fig. 1, azido-5-HETE 3(retention time �12.5 min) was converted to one major metabolite absorbing at 280 nm with aretention time of about 17 min, as expected for azido-5-oxo-ETE 4. The retention time of thissubstance was identical to that of the authentic compound prepared by chemical oxidation(Dess±Martin) of azido-5-HETE. Under the conditions employed, most of the substrate wasoxidized, indicating that the azido derivative 26 is a very good substrate for 5h-dh, and suggestingthat the 125I-labeled compound can be used as a photoa�nity probe to label the enzyme.Preliminary studies now underway suggest that azido-5-HETE is nearly as good a substrate as

Figure 1.

6316

5-HETE for 5h-dh, whereas 5-HETE methyl ester, in which the carboxyl end of the molecule isaltered, is a very poor substrate. These studies will be extended in the near future.The studies reported here support the feasibility of radiolabeling 5h-dh with the 125I version of

26 and this will be our next goal.

Acknowledgements

We wish to acknowledge NIH support under Grants DK-44730 (J.R.), NSF for an AMX-360NMR instrument (Grant CHE-90-13145), and MRC of Canada under Grant MT-6254 (W.S.P.).

References

1. Powell, W. S.; Chung, D.; Gravel, S. J. Immunol. 1995, 154, 4123±4132.

2. O'Flaherty, J. T.; Kuroki, M.; Nixon, A. B.; Wijkander, J.; Yee, E.; Lee, S. L.; Smitherman, P. K.; Wykle, R. L.;Daniel, L. W. J. Immunol. 1996, 157, 336±342.

3. Khanapure, S. P.; Shi, X. X.; Powell, W. S.; Rokach, J. J. Org. Chem. 1998, 63, 337±342.4. Stamatiou, P.; Hamid, Q.; Taha, R.; Yu, W.; Issekutz, T. B.; Rokach, J.; Khanapure, S. P.; Powell, W. S. J. Clin.

Invest. 1998, 102, 2165±2172.5. Powell, W. S.; Gravel, S.; MacLeod, R. J.; Mills, E.; Hashe®, M. J. Biol. Chem. 1993, 268, 9280±9286.6. Norgauer, J.; Barbisch, M.; Czech, W.; Pareigis, J.; Schwenk, U.; Schroder, J. M. Eur. J. Biochem. 1996, 236,

1003±1009.7. Powell, W. S.; MacLeod, R.; Gravel, S.; Gravelle, F.; Bhakar, A. J. Immunol. 1996, 156, 336±342.8. O'Flaherty, J. T.; Taylor, J. S.; Thomas, M. J. J. Biol. Chem. 1998, 273, 32535±32541.

9. Powell, W. S.; Gravelle, F.; Gravel, S. J. Biol. Chem. 1992, 267, 19233±19241.10. Berhane, K.; Ray, A. A.; Khanapure, S. P.; Rokach, J.; Powell, W. S. J. Biol. Chem. 1998, 273, 20951±20959.11. The NMR data for compound 26: 1H NMR (360 MHz, CDCl3) � 8.44 (d, J=2.04 Hz, 1H), 7.78 (dd, J=8.42 and

2.01 Hz, 1H), 7.48 (br s, 1H), 6.95 (d, J=8.4 Hz, 1H), 6.54 (dd, J=15.0 and 11.25 Hz, 1H), 5.98 (t, 10.9 Hz, 1H),5.68 (dd, J=15.1 and 6.6 Hz, 1H), 5.43±5.34 (m, 3H), 4.20 (m, 1H), 3.44 (q, J=6.9 Hz, 2H), 2.94 (m, 2H), 2.39 (m,2H), 2.06 (m, 2H), 1.73 (m, 2H), 1.62 (m, 4H), 1.32 (m, 12H).

12. Perrier, H.; Prasit, P.; Wang, Z. Tetrahedron Lett. 1994, 35, 1501±1502.

13. Mais, D. E.; Yoakim, C.; Guindon, Y.; Gillard, J. W.; Rokach, J.; Halushka, P. V. Biochim. Biophys. Acta 1989,1012, 184±190.

14. Khanapure, S. P.; Saha, G.; Powell, W. S.; Rokach, J. Tetrahedron Lett. 2000, 41, 5807±5811.

6317