J. Med. Chem. 1986, 29, 2351-2358
`
`2351
`
`agent 1 h prior to the injection of carrageenan into one of the hind
`paws. The rats were sacrificed 4 h after administration of the
`drug, at which time both of the hind paws were excised and
`weighed separately. The potencies of the test agents relative to
`phenylbutazone were determined from dose-response plots of the
`the nonin-
`percent increase in weight of the inflamed paw over
`flamed paw. Usually at least three doses, using 6 rats/dose, were
`employed in constructing the plots.
`(c) Inhibition of Phenylquinone-Induced Writhing, The
`assay was performed according to the procedure described in a
`publication23 from these laboratories. Thus, 18-20-g male mice
`were given the test substance orally 20 min prior to an intra-
`peritoneal injection of phenylquinone. The mice were observed
`for the next 10 min for writhing, and the potencies, relative to
`aspirin, were determined as in (b) using 8-10 mice/dose.
`
`Acknowledgment. We thank W. H. Rooks II and A1
`Tomolonis for providing the antiinflammatory and anal-
`gesic data and D. M. Green for the synthesis of some of
`the intermediates.
`
`1 (Rj = OCH3, X = CH2, R2 = H, methyl ester),
`Registry No.
`104156-24-3; la, 54225-54-6; lb, 104156-09-4; lc, 72568-22-0; Id,
`104156-10-7; le, 104156-11-8; le (ethyl ester), 104156-27-6; le (Rj
`le (diazo ketone), 104156-26-5; If,
`= 7-OCH3), 104156-28-7;
`104156-12-9; lg, 55689-65-1; lh, 61220-69-7; li, 104156-13-0; 2a,
`58452-78-1; 2b, 58452-80-5; 2c, 72568-23-1; 2d, 104156-14-1; 3,
`104156-15-2; 5a, 104156-16-3; 5b, 104156-17-4; 6a, 104156-31-2;
`isomer 2),
`isomer 1), 104156-29-8; 6a (stilbene,
`6a (stilbene,
`104156-30-1; 6b, 104156-22-1; 6b (stilbene), 104156-20-9; 6b (R2
`= CH3), 104156-21-0; 6b (diacid chloride), 104156-23-2; 6c,
`2-
`104156-18-5; 3-0HCC6H4CH2C02H-Li, 104156-19-6;
`H3C02CC6H4CH2Br, 2417-73-4; 2-H3C02CC6H4CH2PPh3+Br,
`60494-73-7; 2-H0C6H4CH2C02CH2CH3, 41873-65-8;
`2-
`(2-carbomethoxy-3-methoxy ben-
`H3CC6H4C02CH3, 89-71-4;
`zyl)triphenylphosphonium bromide, 104172-27-2; methyl 2-
`methoxy-6-methylbenzoate, 79383-44-1; 7-methoxy-10,ll-di-
`hydro-5R-dibenzo[a,d]cyclohepten-5-one-2-carboxylic acid,
`64717-08-4; 7-methoxy-10,ll-dihydro-5fi-dibenzo[a,d]cyclo-
`hepten-5-one-2-carboxylic acid, 104156-25-4; 8-methoxy-10,ll-
`dihydro-57f-dibenzo[o,d]cyclohepten-5-one-2-carboxylic acid,
`64453-89-0; aldose reductase, 9028-31-3.
`
`Furanose-Pyranose Isomerization of Reduced Pyrimidine and Cyclic Urea
`Ribosides
`
`HO
`
`OH
`
`James A. Kelley,* * John S. Driscoll,* John J. McCormack,* Jeri S. Roth,* and Victor E. Marquez**
`Laboratory of Medicinal Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer
`Institute, National Institutes of Health, Bethesda, Maryland 20892, and Department of Pharmacology, College of Medicine,
`and Vermont Regional Cancer Center, University of Vermont, Burlington, Vermont 05401. Received December 11, 1985
`Tetrahydrouridine (THU, 2) and other fully reduced cyclic urea
`ribofuranosyl nucleosides undergo a rapid, acid-
`catalyzed isomerization to their more stable ribopyranosyl form. This isomerization is characterized by a change
`in spectral properties and by a greater than 10-fold decrease in potency for those nucleosides that act as potent
`inhibitors of cytidine deaminase in their ribofuranose form.
`l-(/3-D-Ribopyranosyl)hexahydropyrimidin-2-one (7)
`was synthesized and used in conjuction with its furanose isomer 6 as a model compound for more extensive  
`and
`13C NMR, mass spectral, and kinetic studies of this isomerization. The 0.4 & upfield shift and 4-Hz increase in the
`JV|2. coupling constant for the pyranose anomeric proton in the   NMR spectrum is indicative of a pyranose >3-0
`conformation in which the aglycon and C-2' and C-4' hydroxyls are equatorial. The mass spectra of trimethylsilylated
`pyranose nucleosides also show a characteristic large shift in the m/z 204-217 abundance and the appearance of
`two new rearrangement ions at M - 133 and M - 206. For furanose 6 the rate of isomerization is pH and temperature
`dependent with pyranose 7 predominating by a factor of 6-9 at equilibrium. At pH 1 and 37 °C, furanose 6 has
`an initial half-life of less than 12 min. Accordingly, this isomerization may explain the observed lack of enhanced
`ara-C levels in studies evaluating the oral administration of an ara-C and THU combination to species with an acidic
`stomach content.
`Efficient in vivo inhibition of cytidine deaminase (CDA)
`has long been sought as a means of improving the thera-
`peutic activity of the antitumor agent ara-C (la) by ov-
`ercoming its rapid deamination to inactive ara-U (lb).1
`The widespread distribution of CDA in many of the body’s
`tissues, among them the liver, kidney, small intestine, and
`this rapid catabolism of ara-C and other
`blood, ensures
`cytidine analogues.2 Moreover, significant CDA activity
`is encountered in human hematopoietic tissue as well as
`in acute and chronic myelogenous leukemia cells.3,4
`One of the most potent and well-studied CDA inhibitors
`has been tetrahydrouridine (THU, 2).5 However, use of
`THU in combination with ara-C, both in animals and
`humans, has produced disappointing results for the most
`part. There has been little therapeutic advantage observed
`for this combination because of the parallel increase in
`toxicity associated with the resulting high plasma levels
`of ara-C.6,7
`Use of intraperitoneal (ip) combinations of THU and
`ara-C against several in vivo mouse
`tumors, which were
`selected for their high levels of CDA activity, produced a
`
`la, X = NH2
`b, X = OH
`significant increase in lifespan (ILS) against only the as-
`cites form of S180J cells.8 Results with other tumor lines
`
`2
`
`(1) For a recent review, see: Marquez, V. E. In Developments in
`Cancer Chemotherapy·, Glazer, R. I., Ed.; CRC: Boca Raton,
`FL, 1984; pp 91-114.
`(2) Caminier, G. W.; Smith, C. G. Biochem. Pharmacol. 1965, 14,
`1405.
`(3) Ho, D. H. W. Cancer Res. 1973, 33, 2816.
`(4) Fanucchi, M.; Phillips, F.; Chou, T. C. Proc. Am. Assoc. Cancer
`* National Institutes of Health.
`Res. 1984, 25, 20.
`* University of Vermont.
`(5) Hanze, A. R. J. Am. Chem. Soc. 1967, 89, 6720.
`This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society
`
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`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
`
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`

`2352 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 11
`
`Kelley et al.
`
`Table I. Anomeric Proton Signals of the Starting /3-D-Ribofuranosides (RF) and the Corresponding /3-D-Ribopyranosides (RP) Formed
`After Acid Catalysis
`
`B-R
`
`H-V
`
`Jl'2't Hz
`
`compd
`
`R
`
`RP
`
`RP
`
`RP
`
`RP
`
`H-V
`
`t/\'2'i Hz
`
`10.0
`
`10.0
`
`9.6
`
`10.5
`
`h-
`
`5.40
`
`5.00
`
`5.40
`
`5.10
`
`3
`
`5
`
`7
`
`9
`
`B
`
`compd
`
`OH
`\
`
`r
`
`r
`,— NHCx
`
` ^^  
`
`ML/
`HOi_(\h
`
`2
`
`4
`
`6
`
`8
`
`10
`
`R
`
`RF
`
`RF
`
`RF
`
`RF
`
`RF
`
`h
`
`5.80
`
`5.40
`
`5.80
`
`5.40
`
`5.40
`
`6.0
`
`6.0
`
`6.0
`
`7.0
`
`7.0
`
`possessing comparable or higher CDA levels were negative.8
`The only study that reported a therapeutic advantage for
`the above combination over ara-C alone was that of Neil
`et al., where both drugs were administered orally (po) to
`In the same study ip adminis-
`L1210 leukemic mice.9
`tration of both drugs produced no advantage over
`ip ara-C
`alone, possibly because L1210 cells lack CDA activity.
`Thus, it was hypothesized that the inhibition of intestinal
`and bacterial CDA in the lumen permitted a more regular
`and reproducible absorption of ara-C from the gastroin-
`testinal (GI) tract.9’10 A significant increase in therapeutic
`index was observed when the oral combination was given
`either as a single dose or on a daily X 5 schedule.9 These
`two schedules using the oral combination produced ILS
`values of 81% (31% with po ara-C alone) and 130% (50%
`it was
`with po ara-C alone), respectively.9 In some cases
`found that the therapeutic results with the po com-
`even
`bination were superior to those obtained with ip ora-C by
`itself.9 The unique feature of the oral combination was
`the maintenance of lower but effective therapeutic plasma
`levels of ara-C (1 pg/mL) for a longer period of time than
`with the administration of ip aro-C alone.9 Since ara-C
`is a cell-cycle phase-specific agent, the length of time to
`which tumor cells are exposed to a minimum effective drug
`level is of paramount importance. Also effective, but to
`a somewhat lesser degree, was the addition of THU to oral
`5-azacytidine therapy.11
`Despite such encouraging results with the oral combi-
`nation in mice, similar studies in monkeys and humans
`have been very discouraging.6·710 In monkeys, simulta-
`neous oral doses of THU up to 1 g/kg failed to increase
`levels of ara-C given orally at 50 mg/kg.6,10
`the serum
`
`(6) El Dareer, S. M.; Mulligan, L. T., Jr.; White, V.; Tillery, K.;
`Mellet, L. B.; Hill, D. L. Cancer Treat. Rep. 1977, 61, 395.
`(7) Wong, P. P.; Currie, V. E., Mackey, R. W.; Krakoff, I. H.; Tan,
`C. T. C.; Burchenal, J. H.; Young, C. W. Cancer Treat. Rep.
`1979, 63, 1245.
`(8) Kreis, W.; Hession, C.; Soricelli, A.; Scully, K. Cancer Treat.
`Rep. 1977, 61, 1355.
`(9) Neil, G. L.; Moxley, T. E.; Manak. R. C. Cancer Res. 1970, 30,
`2166.
`(10) Kreis, W.; Woodcock, T. M.; Gordon, C. S.; Krakoff, I. H.
`Cancer Treat. Rep. 1977, 61, 1347.
`(11) Neil, G. L.; Moxley, T. E.; Kuentzel, S. L.; Manak, R. C.;
`Hanka, L. J. Cancer Chemother. Rep. 1975, 59, 459.
`
`11
`
`RP
`
`5.10
`
`10.0
`
`Later, when more accurate measurements were made using
`orally administered [14C]THU in humans, the blood levels
`of the drug measured by an enzymatic assay indicated that
`only 23% of the radioactive species in plasma retained
`CDA inhibitory activity.12 No explanation was given to
`account for this result except for the formation of an in-
`active, but uncharacterized, THU dimeric species.10
`We report here the existence of an acid-catalyzed re-
`arrangement and inactivation of THU and several other
`nucleosides that are CDA inhibitors.
`It is possible that
`this pH-dependent rearrangement might account for the
`aforementioned disparate results and the inability of THU
`to enhance oral activity of ara-C in monkeys and humans.
`Chemistry. During the synthesis and evaluation of a
`series of saturated cyclic urea nucleosides (Table I, com-
`pounds 4, 6, 8, and 10), which functioned as very potent
`CDA inhibitors,13 unusual mass spectral and NMR results
`were observed whenever these compounds were exposed
`to acidic conditions or were kept as aqueous solutions for
`extended periods of time. Gas chromatography/mass
`spectrometry (GC/MS) analysis of these nucleosides as
`their per-trimethylsilyl (Me3Si) derivatives did not give the
`anticipated mass spectra,14 although the apparent molec-
`ular weight and degree of trimethylsilylation were as ex-
`pected. Fragment ions derived from the nucleoside sugar
`were not consistent with a ribofuranosyl sugar. The base
`peak was no longer m/z 217; m/z 204 was now a major
`peak, and the M - 103 and m/z 103 peaks, sometimes
`indicative of a 5'-silyloxy moiety,14 were of reduced in-
`In addition, two new peaks appeared at masses
`tensity.
`corresponding to M - 133 and M - 206.
`When D20 solutions of these ribofuranosyl nucleosides
`than 24 h, the
`were kept at room temperature for more
`NMR spectra also showed the progressive appearance of
`a second compound. This newly formed nucleoside ex-
`hibited a different anomeric proton as judged by a change
`in chemical shift as well as coupling constant. This slow
`
`(12) Ho, D. H. W.; Bodey, G. P., Sr.; Hall, S. W.; Benjamin, R. S.;
`Brown, N. S.; Freireich, E. J.; Loo, T. L. J. Clin. Pharmacol.
`1978 18 259
`(13) Liu, P. S.; Marquez, V. E.; Driscoll, J. S.; Fuller, R. W.;
`McCormack, J. J. J. Med. Chem. 1981, 24, 662.
`(14) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.; Town-
`send, L. B.; McCloskey, J. A. J. Org. Chem. 1982, 47, 3923.
`
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`

`

`Furanose-Pyranose Isomerization
`
`Journal of Medicinal Chemistry, 1986, Vol. 29, No. 11
`
`2353
`
`M/Z
`Figure 1. Electron impact mass spectra of (a) per-silylated ribofuranoside 6 and (b) persilylated ribopyranoside 7. Mass spectra are
`the GC peak with background being computer subtracted.
`the weighted average of at least three scans
`over
`transformation in aqueous solution could be dramatically
`accelerated by trace amounts of acid either from a dilute
`HC1 solution or by a few beads of strongly acidic cation
`exchange resin. Under such conditions, NMR spectra
`indicated an equilibrium was established predominantly
`in favor of the newly formed nucleoside. This equilibrium
`mixture was maintained indefinitely and was only slightly
`changed by increased amounts of acid catalyst (vide infra).
`More importantly, when these samples were
`lyophilized
`and tested, CDA inhibitory potencies had dropped several
`orders of magnitude (i.e., K{ >   5 M) when compared to
`the parent compounds (JC¡ = 10'7 to 10*8 M).
`The above mass spectral and NMR observations are
`consistent with an acid-catalyzed ribofuranoside to ribo-
`pyranoside isomerization as depicted in Scheme I. The
`changes in chemical shift and coupling constants of several
`of the starting materials and their rearranged products are
`shown in Table I. These compounds, which include THU
`and other nucleosides with aglycons of varied ring size,
`were originally prepared as CDA inhibitors.13
`The pyranose anomeric proton exhibited an upfield shift
`of 0.3-0.4  , and its
`coupling constant increased by
`ca. 4 Hz relative to that of the furanose. Furthermore, as
`judged from these NMR spectral changes, all the com-
`pounds studied rearranged to the preferred /3-CI pyrano-
`
`side conformation where the glycosidic bond is in an
`equatorial position to allow the H-V and H-2' protons to
`be in the antiperiplanar relationship responsible for the
`large coupling constant observed.15 No other compounds
`were detected by NMR analysis.
`GC/MS analysis of the fully trimethylsilylated /3-d-
`ribofuranosides of Table I and their acid-treated products
`gave both chromatographic and mass spectral evidence of
`a change in molecular structure (Table II). The electron
`impact mass spectrum of the Me3Si derivative of the
`acid-rearranged product dramatically indicated that the
`sugar moiety of the nucleoside had been altered (e.g.,
`Figure lb). As mentioned above, the base peak was no
`longer m/z 217 as expected for a silylated ribofuranoside
`(Figure la), but now m/z 204 was several times more
`in-
`It is well-known that in a six-mem-
`tense than m/z 217.
`bered ring trimethylsilylated sugar, an intense m/z 204
`peak is indicative of multiple vicinal diols.16 Circum-
`
`(15) Townsend, L. B. Synth. Proced. Nucleic Acid Chem. 1973, 2,
`330.
`(16) DeJongh, D. C.; Radford, T.; Hribar, J. D.; Hanessian, S.;
`Bieber, M.; Dawson, G.; Sweeley, C. C. J. Am. Chem. Soc.
`1969, 91, 1728.
`
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`

`

`2354 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 11
`
`Kelley et al.
`
`Table II. Mass Spectral and Gas Chromatographic Data for Trimethylsilylated Ribofuranosyl (RF) and Ribopyranosyl (RP) Nucleosides
`%/“
`
`compd
`uridine
`5,6-dihydrouridine
`2
`3
`4
` 
`6
`7
`8
`9
`10
`11
`“Percent relative intensity.
`
`MW
`no. Me3Si groups
`sugar
`RF
`532
`4
`RF
`534
`4
`RF
`608
`5
`RP
`608
`5
`RF
`506
`4
`RP
`506
`4
`RF
`520
`4
`RP
`520
`4
`RF
`534
`4
`RP
`534
`4
`RF
`622
`5
`RP
` 
`622
`6 Isothermal retention index on an OV-17 liquid phase.
`
`m/z 204
`2.7
`1.1
`1.2
`80
`0.9
`66
`1.1
`76
`2.5
`64
`1.8
`79
`
`m/z 217
`100
`100
`100
`39
`100
`58
`100
`26
`100
`27
`100
`23
`
`RI (OV-17)6
`2610
`2539
`2504
`2581
`2364
`2427
`2413
`2459
`2410
`2471
`2521
`2615
`
`stantial evidence for a pyranose nucleoside also came from
`the reduced relative intensity of the peaks at m/z 103 and
`M - 103, which suggested some modification of the 5'-
`hydroxyl (Me3SiOCH2 after derivatization). That this new
`compound was an isomer was suggested by fragment ions
`indicating that the molecular weight, the degree of sily-
`lation, and the silylated nucleoside base (b) had not
`changed. Mass spectral peaks corresponding to M'+, M
`- CH3, M - Me3SiOH, b + H - CH3, and b + 30 were
`essentially identical to those expected for a trimethyl-
`silylated d-ribofuranosyl nucleoside.14 In addition, two
`new, very characteristic peaks of moderate intensity ap-
`peared at masses corresponding to M - 133 and M - 206.
`In order to verify that this rearrangement was unique
`ring system, uridine and
`for the fully reduced cyclic urea
`treated under the same acidic con-
`dihydrouridine were
`ditions. However, their NMR spectra remained unchanged
`indefinitely. This would suggest that the rearrangement
`through an open-chain Schiffs base, as depicted in Scheme
`I, is favored in the fully reduced compounds by the ability
`of the electrons on the N-l nitrogen to participate in the
`ring-opening process. The aromaticity of the uracil ring
`of uridine prevents this, while the partial double bond
`character between C-2 and N-3 in dihydrouridine must be
`able to provide sufficient conjugative interaction between
`the electron-withdrawing carbonyl at C-4 and N-l
`to ef-
`fectively prevent the ribofuranose to ribopyranose inter-
`conversion. This partial double bond character of amides
`has been recognized as an efficient transmitter of electronic
`and inductive effects.17
`In order to investigate this rearrangement in more detail,
`we selected a model compound on which to perform
`spectral and chromatographic studies. The compound
`chosen was
`l-(j3-D-ribofuranosyl)hexahydropyrimidin-2-one
`(6), which had been prepared previously and was known
`to act as a moderate inhibitor of CDA.5,13 The reason
`for
`selecting compound 6 rather than THU was the absence
`of the unstable carbinolamine moiety found in the latter
`compound. Thus, compound 6 permitted the study of the
`ribofuranose-ribopyranose rearrangement free of other
`possible carbinolamine-related reactions. The study was
`divided into three parts:
`first, chemical synthesis and
`characterization of the authentic rearranged product, 1-
`(/3-D-ribopyranosyl)hexahydropyrimidin-2-one (7); second,
`a detailed NMR and MS study of both starting and re-
`arranged products; and third, a preliminary kinetic in-
`vestigation of the pH and temperature dependence of this
`rearrangement.
`
`Scheme II
`
`Synthesis of Authentic Ribopyranoside.
`1-(/3-d-
`Ribopyranosyl)hexahydropyrimidin-2-one (7) was prepared
`by the Verbruggen modification of a method developed
`earlier by Holy et al.18,19 As shown in Scheme II, the
`2-trimethylsilyloxypyrimidine (13) was condensed with
`in the presence of
`tetra-O-acetyl-D-ribopyranose (12)
`SnCl4.20 The protected ribopyranoside 14 was obtained
`in 70% yield, and the correspondingly deblocked 15 proved
`to be identical in every respect to the compound previously
`reported by Holy et al.18 The remaining step was the facile
`reduction of
`the aglycon to the desired hexahydro-
`pyrimidin-2-one ribopyranoside (7). This compound was
`fully characterized by  
`and 13C NMR, IR, MS, and el-
`emental analysis. As expected, 7 proved to be identical
`to the material isolated from 6 after acid treatment. The
`identity of compound 7 with the isolated acid rearrange-
`ment product was also confirmed by HPLC analysis of a
`mixture of the two compounds.
`Spectral Studies.
`In the high-resolution (250 MHz)
`proton NMR spectrum of 7, the magnitude of the vicinal
`coupling constant of the anomeric proton indicated that
`this compound was the ß isomer.15 The anomeric coupling
`constant obtained for the starting ribofuranose 6 (Table
`I) was compatible with either a or ß configurations. An
`
`(17)
`
`Idoux, J. P.; Kiefer, G. E.; Baker, G. R.; Puckett, W. E.;
`Spence, F. J., Jr.; Simmons, K. S.; Constant, R. B.; Watlock,
`D. J.; Fuhrman, S. L. J. Org. Chem. 1980, 45, 411.
`
`(18) Pischel, H.; Holy, A.; Wagner, G. Collect. Czech. Chem. Com-
`1972, 37, 3475.
`mun.
`(19) Holy, A. Collect. Czech. Chem. Commun. 1977, 42, 902.
`(20) Neidballa, V.; Vorbruggen, H. J. Org. Chem. 1974, 39, 3668.
`
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`

`Furanose-Pyranose Isomerization
`
`Journal of Medicinal Chemistry, 1986, Vol. 29, No. 11
`
`2355
`
`Table III.
`
`13C NMR Spectra of Ribofuranoside (6) and Ribopyranoside (7) Nucleosides of Hexahydropyrimidin-2-one
`4
`4
`
`carbons, ppm
`2'
`3'
`2“
`1'
`compd
`6
`5
`87.69, d
`22.03, t
`70.48, d
`69.90, d
`b
`155.27, a
`b
`6
`71.82, d
`79.53, d
`22.07, t
`67.30, d
`b
`7
`b
`156, s
`“Multiplicity indicated below the value for 13C chemical shifts. 6Obscured by solvent (Me2SO-d6).
`
`4
`
`4'
`82.71, d
`66.53, d
`
`5'
`62.00, t
`64.73, t
`
`Table IV. Major Ions from Trimethylsilyated
`l-(/3-D-Ribopyranosyl)hexahydropyrimidin-2-one (7)
`
`ions
`
`M-+
`m-ch3
`M - Me3SiOH
` 
`- Me3SiOCHCH2OH
`S-H
`M - C2H402(Me3Si)2
`C3H302(Me3Si)2
`C2H202(Me3Si)2
`b + H - CH3
`Me3SiO+=Si(CH3)2
`(CH3)3Si
`
`mass,
`Me3Si-d„
`520
`505
`430
`387
`348
`314
`217
`204
`157
`147
`73
`
`m/z
`Me3Si-d9
`556
`538
`457
`414
`375
`332
`235
`222
`163
`162
`82
`
`no. Si
`4
`4
`3
`3
`3
`2
`2
`2
`1
`2
`1
`
`obsd
`mass,
`m/z
`520.260
`505.232
`430.209
`387.191
`348.157
`314.145
`217.099
`204.091
`157.074
`147.057
`73.050
`
`unequivocal chemical proof of the ß configuration for this
`compound, however, was performed earlier in this labo-
`ratory.13
`The 13C NMR spectral assignments listed in Table III
`for carbons 5 and 5' were based on the observed off-reso-
`nance coupling patterns. The assignments for C-T were
`based on literature data for similar compounds, which
`predicts that the anomeric carbon is the lowest field carbon
`in the sugar group.21 Carbon 2 was distinguished by its
`intensity, indicative of the low nuclear Overhauser effect
`on the carbonyl carbon. Carbons 2' and 3' were assigned
`by comparison with the data of Gorin who used specifically
`labeled deuterated sugars.22 The C-l/ carbon resonance
`was shifted upfield 8.16 ppm in ribopyranose 7, relative
`to ribofuranose 6. Likewise, the upfield shift (16.18 ppm)
`of the C-4' absorbance in 7, as compared to that in 6, is
`another consequence of the sugar ring expansion, which
`the cyclic ether oxygen away from this carbon atom.
`moves
`These upfield shifts appear to be the salient characteristics
`in the transformation of 6 to 7 as observed in the 13C NMR
`spectrum.
`Additional mass spectral studies were also undertaken
`in order to characterize further the M - 133 and M - 206
`fragment ions and to confirm assignments based on nom-
`inal mass data. Trimethylsilylation with bis(trimethyl-
`silyl)acetamide-dlg was used to determine the extent of
`silylation and the number of Me3Si groups in each frag-
`ion (Table IV). Accurate mass measurement was
`ment
`also employed to check the tentative structural assign-
`ments for the major fragment ions observed in the mass
`
`(21) Jones, A. J.; Grant, D. M.; Winkely, M. W.; Robins, R. K. J.
`Phys. Chem. 1970, 74, 2684.
`(22) Gorin, P. A. J.; Mazurek, M. Can. J. Chem. 1975, 53, 1212.
`
`Table V.
`Isomerization Kinetics of Ribofuranose 6
`apparent ty3,a h
`apparent pH
`temp, 0 C
`fi*,6
`<0.2
`0.12
`1.0
`25
`2.23 ± 0.05
`2.0
`0.11
`25
`0.63
`2.0
`0.18
`37
`5.2 ± 0.3
`0.16
`37
`2.8°
`the initial pseudo-first-order
`0 Half-life of
`isomerization.
`6 Apparent ratio of 6 to 7 at equilibrium.
`“0.1 M acetic acid.
`
`Time (h)
`Figure 2. The furanose-pyranose isomerization of model com-
`pound 6 in pH 1 HC1 buffer at 25 °C. Mixture components were
`separated by reverse-phase HPLC and monitored at 215 nm (see
`(O) ribofuranoside 6, ( ) ribo-
`Experimental Section). Key:
`pyranoside 7, (A) ribopyranoside 16a.
`spectrum of silylated ribopyranose 7 (Figure lb, Table IV).
`The M - 133 fragment (m/z 387) represents the loss of
`C2H302SiMe3 from the molecular ion, while M - 206 (m/z
`314) is an odd-electron ion resulting from the formal loss
`of C2H402(SiMe3)2. Both of these ions apparently arise
`by fragmentation of the silylated pyranose ring and they
`are, for all intents and purposes, absent (relative abun-
`dances less than 0.2% and not the most
`intense peak in
`a cluster) in silylated furanose isomer 6. Similar frag-
`mentation patterns are observed in the mass spectra of the
`
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`

`2356 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 11
`
`Table VI. CDA Inhibitory Activity of Ribofuranoside and
`Ribopyranoside Forms of Various Cyclic Urea Nucleoside
`Inhibitors
`
`K¡, M, mouse kidney CDA
`1.5 x 10"7
`2 X 10"5
`4 X 10-*
`>5 X 10-5
`3 X 10"7
`6.6 x 10"*
`ribofuranose and ribopyranose
`
`compd pairs
`2 (ribofuranose)
`3“ (ribopyranose)
`6 (ribofuranose)
`7 (ribopyranose)
`8 (ribofuranose)
`9“ (ribopyranose)
`“Results from a mixture of
`formed after acid treatment.
`trimethylsilyl derivatives of pyranoses 3, 5, 9, and 11.
`Kinetics. The rate of isomerization of ribofuranose 6
`to ribopyranose 7 as a function of pH was determined by
`HPLC analysis of solutions of 6 buffered in the pH 1-3
`range and maintained at either 25 or 37 °C. As expected,
`lowering the pH or raising the temperature accelerated the
`rate of isomerization (Table V). At pH 1 and 25 °C the
`so rapid that
`rate of isomerization was
`the apparent
`half-life for 6 could not be accurately determined (Figure
`2). However, at pH 2 and 25 °C the disapperance of 6
`with respect to time initially followed pseudo-first- order
`In all
`kinetics with an apparent half-life of 2.23 ± 0.05 h.
`cases a constant ratio of 6 to 7 was eventually reached with
`the ribopyranose form predominating by factors of 6-9.
`That this isomerization results in an equilibrium was
`further demonstrated by facile conversion of synthetic 7
`to 6 at pH 1 and 25 °C to give an identical ratio of 6 to
`7 as that observed in Figure 2.
`The slow increase of an additional, longer retained peak
`with a k' = 5.16 was observed during HPLC analysis of 6
`and 7 in acid buffer (dashed line, Figure 2). The formation
`of this material may account for the gradual and parallel
`decrease in both 6 and 7; however, nothing can be said
`about the relative amount of this compound since its ex-
`tinction coefficient at 215 nm is not known. Semiprepa-
`rative HPLC was used to isolate this material for further
`characterization by NMR and mass spectrometry. Proton
`NMR indicated a nucleoside with the anomeric proton
`being a doublet centered at 5.55   and with Jv¡2> equal to
`10 Hz. The positive ion FAB mass spectrum showed an
`apparent protonated molecular ion at m/z 263, indicating
`an increase of 30 daltons over either 6 or 7. GC/MS
`analysis of this material after trimethylsilylation was es-
`pecially revealing. A ribopyranose sugar was clearly in-
`dicated by the m/z 204 base peak and other diagnostic ions
`(e.g., M - 133, M - 206, S - H at m/z 348) in the electron
`impact spectrum. The site of modification was identified
`as the nucleoside base, which now possessed a moiety that
`could be readily trimethylsilylated and was 30 daltons
`than the base in 6 or 7. Based on this evidence, this
`more
`derivative was
`tenatively assigned structure 16b. Thus,
`compound 16a probably arises from reductive N-
`formylation of 7 because of the 200 ppm formaldehyde that
`is added to the commerical buffer as a preservative.
`
`^^*<^   -  2  
`
`RO
`
`16a, R = H
`b, R = Si(CH3)3
`Biology. As observed in Table VI, CDA inhibitory
`activity was dramatically reduced for the ribopyranoside
`nucleosides. This was particulary noteworthy in the case
`In the
`of compound 7, which was studied in its pure form.
`
`Kelley et al.
`other cases
`(i.e., compounds 3 and 9) the assay mixtures
`obtained after acid treatment contained greater than 90%
`of the ribopyranoside form as evidenced from NMR.
`These samples were obtained from the aqueous solutions
`of the pure ribofuranosides after treatment with excess
`acidic resin for 15 min and subsequent lyophilization.
`It
`is possible that the residual ribofuranoside contributed a
`significant portion of the activity seen in these equilibrium
`mixtures.
`Discussion
`Ribofuranoside to ribopyranoside rearrangements have
`been described previously. The very simple l-(/3-D-ribo-
`furanosyl)urea has been reported to convert slowly to the
`ribopyranoside form in aqueous solution at room tem-
`perature.23,24 Likewise, a furanoid glycosyl derivative of
`5,5-dibromo-5,6-dihydro-6-hydroxyuracil underwent
`anomerization as well as ring expansion of the sugar moiety
`to give the corresponding pyranoside form.25 Some ami-
`nopyrimidine nucleosides, derived from the opening of the
`imidazole ring in certain purines, also underwent rear-
`rangement to the more stable ribopyranoside form after
`acid treatment.26 Finally, the deblocking of N-(2',3',5'-
`tri-O-acetylribofuranosyl) anthranilonitrile with methanolic
`ammonia resulted in the formation of the more
`thermo-
`dynamically favored ß-pyranoside isomer.27
`The THU literature does not address the problem of
`ribofuranose to ribopyranose rearrangement in the cases
`Information re-
`where this drug has been given orally.
`garding the acid stability of THU, however, was reported
`by chemists at Stanford Research Institute who analyzed
`different lots of THU for the NCI. They considered the
`material to be highly unstable under acidic conditions as
`judged by NMR and optical rotation measurements.28 At
`that time, however, it was not realized that there was a
`concomitant loss of biological potency associated with the
`acid instability of the drug. This finding is of particular
`importance when one observes that patients who received
`oral THU had fasted for 12-18 h and that the solution of
`the drug was often given with an equal volume of orange
`juice. Human gastric fluid has a pH of 1.2-1.7s9 and orange
`juice can be as low as pH 3.30 This combination of cir-
`cumstances makes it very likely that THU was being
`rapidly inactivated and therefore rendered ineffective in
`protecting ara-C from deamination. This would also ex-
`plain the discrepancy between the radioactive and enzy-
`matic assays in determining THU plasma levels.12 Since
`the study of Neil et al.9 showed a definite advantage with
`orally administered THU in mice, while the work of Kreis10
`in humans and El Dareer6 in monkeys did not, it is possible
`in light of the present discussion that this is simply the
`result of pH differences between the stomachs of the ex-
`In agreement with this suggestion,
`perimental subjects.
`the reported values for the pH of the stomach contents in
`
`(23) Ukita, T.; Hamada, A.; Yoshida, M. Chem. Pharm. Bull. 1964,
`12, 454.
`(24) Naito, T.; Kawakami, T. Chem. Pharm. Bull. 1962, 10, 627.
`(25) Cadet, J. In Nucleic Acid Chemistry; Townsend, L. B., Tipson,
`R. S., Eds.; Wiley: New York, 1978; Part I, pp 311-315.
`(26) Montgomery, J. A.; Thomas, H. J. J. Org. Chem. 1971, 36,
`1962.
`(27) Ferris, J. P.; Singh, S.; Newton, T. A. J. Org. Chem. 1979, 44,
`173.
`(28) Giampietro, S.; Cheung, A.; Lim, P. Stanford Research Insti-
`tute Report, Contract NOl-CM-33723, Dec. 3, 1975.
`(29) Documenta Geigy, 6th ed.; Diem, K., Ed.; Geigy Pharmaceu-
`ticals: Ardsley, NY, 1962; pp 520-523.
`(30) Handbook of Chemistry and Physics, 62nd ed.; Weast, R. C.,
`Astle, M. J., Eds.; CRC:Boca Raton, FL, 1981; p D-127.
`
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`Petitioner Apotex Exhibit 1015-0006
`
`

`

`Furanose-Pyranose Isomerization
`mice fluctuates between 2.2 and 4.8.31 Since the acid
`stability and the kinetics of isomerization of THU are
`similar to 6,32 THU in its active ribofuranose form would
`have been inactivated to a great extent during oral ad-
`ministration to humans.
`It can be concluded, therefore, that stomach acidity
`might be a determining factor in controlling the concen-
`tration of THU that is available to block deamination of
`ara-C in the GI tract. As discussed earlier, inhibition of
`the deamination reaction is an important prerequisite for
`a gradual absorption of ara-C. This situation has probably
`never been achieved in the animal experiments or during
`the THU clinical trials where ara-C and THU were used
`concomitantly in species with an acidic stomach content.
`Experimental Section
`General Methods.   NMR spectra for routine samples were
`recorded on Varían T-60 instrument. For high resolution, a
`Brucher WM 250 model was used. High-resolution 1H NMR and
`IR
`13C NMR studies were performed using Me2SO-d6 as solvent.
`spectra were obtained on a Perkin-Elmer 727B infrared spec-
`trophotometer with the samples as Nujol mulls. Melting points
`were determined on a Thomas-Hoover apparatus and are un-
`corrected. Elemental analyses were performed by Galbraith
`Inc., Knoxville, TN. Cytidine deaminase was
`Laboratories,
`kidney preparation with the same
`measured by using a mouse
`methodology as reported previously by us.13
`Microscale trimethylsilylation of nucleosides (1-2 mg) and the
`corresponding isomerized mixtures was conducted at room tem-
`perature with 0.45 mL of a 1:2 (v/v) solution of bis(trimethyl-
`silyl)trifluoroacetamide (BSTFA) and redistilled acetonitrile. The
`trimethylsilyl-dg derivatives of compounds 6 and 7 were also
`prepared. About 1 mg of each isomer was individually reacted
`with 0.15 mL of a 1:2 (v/v) mixture of bis(trimethylsilyl-d9)-
`acetamide and redistille