A novel and efficient asymmetric synthesis of carbon-14 labeled (S, S)-2,7-di-boc-diamino[1,8-14C2]suberic acid

Article abstract of DOI:10.1002/jlcr.534

Five hundred mCi of Potassium [¹⁴C]cyanide at a specific activity of 51 mCi/mmol was used to diastereoselectively introduce the carbon-14 label into 1,6-hexanedial via a thermodynamically controlled asymmetric Strecker reaction using (R)-(-)-2-phenylglycinol as the chiral auxiliary. The expected and predominant (R, S/S, R) diastereomer (2) was separated by preparative normal phase HPLC. The chiral auxiliary was removed by oxidation with lead tetraacetate and the resulting benzylimino nitrile exhaustively hydrolyzed in hydrochloric acid to give (S,S)-2,7-diamino[1,8-¹⁴C₂]suberic acid (6). Subsequent acylation with di-tert-butyldicarbonate gave the title compound (1) with a radiochemical purity of 95,5%, a specific activity of 113 mCi/mmol, and an enantiomeric purity of 95.5% e.e. To our knowledge this is the first report of the asymmetric Strecker methodology being applied to the synthesis of a carbon-14 labeled amino acid. Copyright

Full text of DOI:10.1002/jlcr.534

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2002; 45: 49–57.
DOI: 10.1002/jlcr.534
Research Article
A novel and efficient asymmetric synthesis of
carbon-14 labeled (S; S)-2,7-di-boc-
diamino[1,8-14C2]suberic acid
A. J. Villani*, D. Saunders, A. Y. L. Shu and J. R. Heys
GlaxoSmithKline Pharmaceuticals, Department of Process Strategy and Support,
Isotope Chemistry Section, UW2830, King of Prussia, PA 19406, USA
Summary
Five hundred mCi of Potassium [14C]cyanide at a specific activity of 51 mCi/
mmol was used to diastereoselectively introduce the carbon-14 label into 1,6-
hexanedial via a thermodynamically controlled asymmetric Strecker reaction
using (R)-(-)-2-phenylglycinol as the chiral auxiliary. The expected and
predominant (R; S=S; R) diastereomer (2) was separated by preparative normal
phase HPLC. The chiralauxial ry was removed by oxidation with elad
tetraacetate and the resulting benzylimino nitrile exhaustively hydrolyzed in
hydrochloric acid to give (S; S)-2,7-diamino[1,8-14C2]suberic acid (6). Subse-
quent acylation with di-tert-butyldicarbonate gave the title compound (1) with
a radiochemicalpurity of 95.5%, a specific activity of 113 mCi/mmo,l and an
enantiomeric purity of 95.5% e.e. To our knowledge this is the first report of
the asymmetric Strecker methodology being applied to the synthesis of a
carbon-14 labeled amino acid. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: asymmetric Strecker reaction; thermodynamic control; (R)-(-)-2-
phenylglycinol chiral auxiliary; potassium [14C]cyanide; carbon-14 labeled
amino acid
*Correspondence to: A. J. Villani, GlaxoSmithKline Pharmaceuticals, Department of Process
Strategy and Support, Isotope Chemistry Section, UW2830, King of Prussia, PA 19406, USA.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 16 February 2001
Revised 1 October 2001
Accepted 3 October 2001

50
A. J. VILLANI ET AL.
Introduction
Peptides in which the disulfide linkage of the cystine unit has been
replaced with the isosteric dicarba equivalent 1 have shown high
biological activity and enhanced metabolic and chemical stability.1 As a
result, interest in the synthesis of 1 has gained much attention in the last
few years.2,3
The peptide SK&F-107647 was selected from preliminary screens for
further study because of its potency as a regulator of CSFs (colony
stimulating factors) in certain cell lines.4,5 To determine the safety of
SK&F-107647 in preclinical species, whole body autoradiography
(WBAR) and excretion studies in rats and dogs were needed using
isotopically labeled peptide. Since the studies with tritium labeled
peptide did not provide definitive data due to exchange with water in
vivo, a high specific activity carbon-14 labeled analog of SK&F-107647
was requested with the carbon-14 located in the more metabolically
stable part of the molecule, namely, the isosteric dicarba equivalent of
cystine (Figure 1).
This necessitated the synthesis of carbon-14 labeled chiral (S; S) 2,7-
Di-BOC-diaminosuberic acid (1, Figure 1) labeled at C1 and C8
described herein. Asterisks denote the positions of the carbon-14 labels.
Results and discussion
Of the various synthetic strategies available to us, the Strecker amino
acid synthesis offered the most convenient and inexpensive experimental
Figure 1. Structure of SK&F-107647 and 1
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[
14C] LABELED (S, S)- SUBERIC ACID
51
protocols for preparing carbon-14 labeled amino acids. The Strecker
reaction has been known since 1850, and the first asymmetric Strecker
reaction was reported in 1963 by Harada.6
The Strecker methodology we chose to follow relied greatly on the
work of Ogura7 (Figure 2) who achieved a high equilibrium ratio of I:II
in a thermodynamically controlled 1,3-asymmetric induction using (R)-
(-)-2-phenylglycinol as the chiral auxiliary. Thus, when the appropriate
aldehyde is treated with (R)-(-)-2-phenylglycinol in the presence of KCN
and NaHSO3 in aqueous methanol an equilibrium is established. This
equilibrium is believed to involve the intermediacy of the Schiff base A.8
We envisaged a similar equilibration in the novel case of a dialdehyde.
Our results obtained with 1,6 hexanedial appear to be consistant with
the above equilibrium hypothesis in that a diasteroselectivity of 3:1 was
obtained in favor of the desired and equivalent (R; S=S; R) isomer (2).
In addition to the use of a dialdehyde, other modifications of the Ogura
procedure7 were necessary in order to conform to the usualradio-
synthetic requirements. In our case, this entailed making potassium
[14C] cyanide the limiting reagent.
The unstable 1,6-hexanedial (2 eq.) was generated in situ via the
oxidation of cyclohexene9 and then treated directly with 500 mCi
(1.6 eq.) of potassium [14C]cyanide in the presence of (R)-(-)-2-
phenylglycinol, giving a 3:1 mixture of diastereomers 2 (R; S=S; R)
and 3 (R; S=R; R) (meso), respectively. Very little of the (R; R=R; R)
isomer (4) was produced. As expected, the (R; S=S; R) diastereomer (2)
was shown to give 1 as determined by chiral capillary electrophoresis
analysis. In non-isotopic trial runs, the above diastereomeric mixture
was carried through to a mixture of enantiomers containing 1, and then
analyzed by chiral capillary electrophoresis versus reference standards
for the (S; S), meso (S; R), and (R; R) isomers of 1. The analysis showed
the enantiomeric mixture to contain: 72% (S; S), 25% meso and 3%
(R; R).
Figure 2. Equilibration of a-aminonitriles derived from (R)-(-)-2-phenylglycinol
and aldehydes
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A. J. VILLANI ET AL.
The predominant and desired diastereomer 2 was separated by
preparative normal phase HPLC in an overall yield of 29% from
potassium [14C]cyanide. Surprisingly, attempts to remove the chiral
auxiliary with Pd-catalyzed hydrogenolysis10 were not successful.
However, we discovered that oxidation of 2 with 3 equivalent of lead
tetraacetate11 in methylene chloride at 08C afforded the Schiff base 5 in
an 86% radiochemical yield. Subsequent hydrolysis of the resulting
crude 5 with 6 N HClat ambient temperature, then with concentrated
HClat 95 8C afforded the expected 6 in an 89% radiochemicalyield. No
racemization was detected by capillary GC.
The ensuing acylation of 6 with di-tert-butyldicarbonate gave a 54%
radiochemicalyield of 1 having a radiochemicalpurity of 95% (TLC), a
specific activity of 113 mCi/mmol(MS), and an enantiomeric purity of
95.5% e.e. (GC) (Scheme 1).
Scheme 1. Synthesis of (S, S)-2,7-Di-BOC-diamino[1,8-14C2]suberic acid (1).
Reagents and conditions: a. OsO4(cat), b. NalO4, Et2O/H2O, rt; c. K*CN,
d. NaHSO4, e. (R)-(-)-2-phenylglycinol, CH3OH/H2O, rt, 68 h; f. LTA, CH2Cl2,
08C; g. 6N HCl, rt, h. cHCl, 958C, 18 h; i. (BOC)2O, pH 9, aq. CH3CN, rt, 14 h
Copyright # 2002 John Wiley & Sons, Ltd.
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[
14C] LABELED (S, S)- SUBERIC ACID
53
Compound 1 was successfully utilized in the synthesis SK&F-107647-
[14C] (Shu AYL, Heys JR. GSK Unpublished Results 1996).
Conclusion
We believe that the generality, mild conditions, convenience and
efficiency of this methodology make it worthy of a place in the arsenal
of carbon-14 chiral amino acid labeling techniques. Hanus12 reported a
racemic synthesis of very low specific activity carbon-14 labeled 6 via the
alkylation of sodium diethylacetamido [2-14C]malonate with 1,4-
dibromobutane. Our method compares favorably to that of Hanus’s
in terms of overall radiochemical yield to 6, and has the added
advantage of using less expensive labeled starting materials.
To our knowledge, this is the first report of 1 being prepared labeled
with carbon-14 via an asymmetric Strecker reaction, and the first
example of Ogura’s methodology being applied to the preparation of a
chiralbis-amino acid.
Experimental section
General
Low-resolution mass spectra were obtained on a Finnegan 3625
instrument operating in the chemicalionization mode using ammonia
as reagent gas for the determination of specific activity. Enantiomeric
purities were obtained on Hewlett-Packard 5890 gas chromatograph
TM
using a ChirasilVal
25 M ꢀ 0.25 mm ꢀ 0.16 um column, injector
temperature of 2508C, detector temperature of 2758C, FID detector,
linear velocity 40 cm/s, carrier gas He, makeup gas N2, injection split
1.5:1, temperature profile: 908C for 4 min then 48C/min to 1808C for
20 min. Thin layer chromatograms were run on 5 ꢀ 20 cm, 250 um
Merck and Analtech Silica Gel plates using a solvent system of 10:1:2
C2H5OH:H2O:15 N NH4OH except where noted in the preparation.
Capillary electrophoresis was carried out on a fused silica, 50 cm (length
to detector), 57 cm (total length), 50 um I.D. column, and BGE
(background electrolyte) of 0.1 M H3PO4 brought to pH with NaOH;
0.025 M hydroxypropylbetacyclodextrin; 5% acetonitrile, 30 kV, 200 nm
detection. HPLC analyses were carried out on a Varian 5000 HPLC
system using a Zorbax SBTM C18 5 um 4.6 mm ꢀ 250 mm column,
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A. J. VILLANI ET AL.
220 nm, 1 ml/min and radioactivity was monitored with a Ramona
Radioactivity Detector with TruCountTM scintillation cocktail in a flow
cell. Potassium [14C]cyanide was purchased from IICH through
Moravek Biochemicals.
1,6-Hexanedial. To a 100 ml, 2-neck round bottom flask fitted with a
thermometer and magnetic stirrer, was added 30 mleach of water and
diethyl ether. To this was added cyclohexene (822 mg, 10 mmol) and
2 ml of a 4% aqueous solution of osmium tetroxide (80 mg, 0.3 mmol).
To the vigorously stirred dark brown mixture was added sodium
periodate (5.25 g, 25 mmol) portionwise at ambient temperature over a
period of 45 min. Occasionalcooinl g with an ice/water bath was
necessary to maintain the internaltemperature at about 25 8C. After
stirring for additional3 h, the ensuing white precipitate (sodium iodate)
was filtered off and the layers separated. The organic phase was
concentrated to dryness in vacuo at ambient temperature and the
resulting dark brown oil used directly in the next step.
(S)-2,7-Bis[(R)-2-hydroxy-1-phenylethylamino]-subero[1,8-14C2]nitrile
(2). To the chilled, dark brown oily 1,6-hexanedial (703 mg, 6.2 mmol)
in a single neck 100 ml round bottom flask was added sequentially: 30 ml
of a chilled aqueous solution of sodium bisulfite (1.28 g, 12.3 mmol),
solid potassium [14C]cyanide (638.6 mg, 9.8 mmol, 500 mCi, SA 51 mCi/
mmol), and 10 ml of a chilled methanolic solution of (R)-(-)-2-
phenylglycinol (1.69 g, 12.3 mmol). A magnetic stirring bar was
introduced, the flask stoppered, and its contents stirred at ambient
temperature for 68 h. The orange colored oily suspension was extracted
with ethylacetate (3 ꢀ 40 ml). The aqueous phase (pH 9.9) was diluted
to 100 mlwith water. The aqueous phase contained 138 mCi,
presumably inorganic, and was discarded.
The ethylacetate extract (362 mCi), containing 66% of the desired
diastereomer (2) by radio-HPLC (60:40 CH3CN:0.1% aqueous TFA),
was concentrated in vacuo to a small volume and placed atop a 20 mm
ID ꢀ 300 mm Silica (Merck 60, 230–400 mesh) flash chromatography
column. The column was eluted with ethyl acetate to give 246 mCi of a
diastereomeric mixture free of polar contaminants. This represents a
49% radiochemicalyiedl from potassium [ 14C]cyanide.
The ethyl acetate eluant (150 ml) was concentrated in vacuo to a
volume of 15 ml, and 15 ꢀ 1 mlinjections made onto a 41.4 mm
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14C] LABELED (S, S)- SUBERIC ACID
55
ID ꢀ 250 mm, Rainin DynamaxTM, 60 A, 8 um Silica column, with UV
detection (254 nm), 40 ml/min flow rate, using a solvent system of 90:10
ethylacetate/hexane. The desired diastereomer eulting at 7 min was
collected and evaporated in vacuo to give 524 mg (146 mCi) of 2 for a
29% radiochemicalyiedl from potassium [ 14C]cyanide. Compound 2
was a single component by chiral GC assay with a retention time of
16 min. A 73 mCi portion of 2 was used directly in the next step. The
1
structure of the non-isotopic form of 2 was supported by 360 MHz H
NMR (CHCl3): dH 1.46 (m, 4H), 1.74 (m 4H), 2.17 (broad s, 4H, OH,
NH), 3.23 (t, 2H), 3.55 (m, 2H), 3.73 (m, 2H), 4.08 (m, 2H), 7.53 (m,
10H).
(S,S)-2,7-Bis[N-benzylideneamino]subero[1,8-14C2]-nitrile (5). To a 100 ml
round bottom flask fitted with an addition funnel, magnetic stirrer,
thermometer and argon inlet, was added methanol (20 ml) and methylene
chloride (10 ml). The flask was purged with argon then placed in a dry ice/
acetone cooling bath and cooled to an internal temperature of 108C. Lead
tetraacetate (851 mg, 1.9 mmol) was weighed out under argon and then
added to the flask in one portion producing a clear, bright, lemon colored
solution. The solution temperature was lowered to 08C and 2 (262 mg,
0.64 mmol, 73 mCi) in methylene chloride (25 ml) added by drops over a
period of 44 min. The color of the reaction remained unchanged during the
addition. After stirring an additional30 min at 0 8C, saturated aqueous
solution of sodium bicarbonate (15 ml) was added. The rapid addition
produced a carbon dioxide discharge and a color change to orange
accompanied by a temperature increase to 108C. The mixture was
transferred to a 250 mlseparatory funneland the alyers separated. The
aqueous layer was extracted with methylene chloride (3 ꢀ 25 ml). The
methylene chloride extracts were combined, washed with saturated
aqueous sodium chloride, and then dried over sodium sulfate. The drying
agent was removed by filtration and the filtrate evaporated to an oil in
vacuo. The oilweighed 188 mg (63 mCi) for a radiochemicalyiedl of 86%,
and was used directly in the next step.
(S,S)-2,7-Diamino[1,8-14C2]suberic acid (6). To 5 (188 mg, 0.55 mmol,
63 mCi) in a 25 mlsingle neck round bottom flask fitted with a magnetic
stirrer was added chilled 6N HCl (9 ml). The mixture was stirred at
ambient temperature for 22 h. The benzaldehyde by-product formed an
oily suspension, which was removed by extraction with ethyl acetate.
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A. J. VILLANI ET AL.
The resulting clear aqueous phase contained 100% of the radioactivity
at Rf ¼ 0:65 by radio-TLC.
The above aqueous solution was concentrated in vacuo (358C/30 mm
Hg) and the resulting residue heated with 12N HCl (10 ml) at 90–958C
for 18 h. A radio-TLC trace showed 95% of the radioactivity was now
at Rf ¼ 0:13. The aqueous solution was evaporated to dryness in vacuo
(358C/30 mm Hg) and the residue treated with 15 N ammonium
hydroxide, then lyophilized to dryness. The resulting solid was
suspended in absolute ethanol, collected by filtration and then dried
in vacuo for 21 h at 468C/30 mm Hg.
The solid weighed 107 mg (56 mCi) for a radiochemical yield of 89%,
and a specific activity of 113 mCi/mmolby MS. The materialwas used
directly in the next step.
Mass spectrum (CI, ammonia, m=z (%): (M+H)+ 0-14C 205 (3),
(M+H)+ 1-14C 207 (28), (M+H)+ 2-14C 209(100).
(S,S)-2,7-di-boc-diamino[1,8-14C2]suberic acid (1). To a 25 mlsingel
neck round bottom flask fitted with a pH probe, magnetic stirrer and an
addition funnelwas added 6 (107 mg, 0.5 mmol, 56 mCi) dissolved in
0.2 M aqueous sodium carbonate (5 ml) and acetonitrile (10 ml). The
reaction mixture (a thin suspension) was cooled in an ice/water bath and
2.5 N aqueous sodium hydroxide added to pH=11, forming a solution.
A solution of di-tert-butyldicarbonate (352 mg, 1.6 mmol) in acetonitrile
(1 ml) was added in one portion. The reaction mixture was stirred in an
unreplenished ice/water cooling bath for 3 h, then at ambient
temperature for 14 h. A pH=9 was maintained during the course of
the reaction by the occasionaladdition of 2.5 N aqueous sodium
hydroxide. A radio-TLC assay showed 4 spots migrating at Rf 0.16
(2.8%, 6), 0.55 (90%, 1) and two unknowns at 0.82 (5.2%) and 0.99
(1.5%).
The reaction mixture was concentrated in vacuo (to remove
acetonitrile), and diluted with water (40 ml). The pH was 10.4. The
aqueous solution was acidified to pH=1.7 with 1 M aqueous potassium
bisulfate. The resulting liberated oil was extracted with ethyl acetate.
The extracts were combined, washed with saturated aqueous sodium
chloride solution, dried briefly over sodium sulfate, filtered, and the
filtrate evaporated to an oil in vacuo at 358C. The oil was crystallized by
trituration with acetonitrile. The solid was collected by filtration and
dried for 41 h at ambient temperature under high vacuum. The product
weighed 120 mg (30 mCi) for a radiochemicalyiedl of 54%. The
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[
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radiochemicalpurity by radio-TLC was 95% ( Rf ¼ 0:68), specific
activity (MS) 113 mCi/mmolwith a 95.5% e.e. ( S; S) enantiomeric
purity by chiralGC.
Mass Spectrum (CI, Ammonia, m=z (%): (M+H)+0-14C, 405 (nil),
(M+H)+1-14C, 407 (13), (M+H)+2-14C, 409 (51).
Acknowledgements
The authors wish to thank Messrs. W.J. Bray and S.H. Levinson for the
radioanalyses, Messrs. L. Killmer and A. Allen for the MS and
[1H]NMR analyses, and Mr. Y. Rawjee for the chiral capillary
electrophoresis data. The authors wish to express a special thanks to
Dr. J. Hieblfor providing the ( S; S), meso, and (R; R)-Di-BOC-
diaminosuberic acid unlabeled reference standards.
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