Synthesis and configuration of the cyclin-dependent kinase inhibitor roscovitine and its enantiomer

Article abstract of DOI:10.1016/S0957-4166(01)00471-2

The cyclin-dependent kinase inhibitor (R)-2-(6-benzylamino-9-isopropyl-9H-purin-2-ylamino)butan-1-ol (roscovitine, 1a), as well as its (S)-enantiomer 1b, were synthesised. The chemical structure and absolute configuration of both enantiomers was confirmed by X-ray crystallography. Furthermore, high enantiomeric excess (>98%) was demonstrated by chiral chromatography of 1a and 1b, as well as NMR analysis of the diastereomeric Mosher's ester derivatives 2a and 2b.

Full text of DOI:10.1016/S0957-4166(01)00471-2

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2891–2894
Synthesis and configuration of the cyclin-dependent kinase
inhibitor roscovitine and its enantiomer
Shudong Wang,a Steven J. McClue,a John R. Ferguson,b Jonathan D. Hull,b Steven Stokes,b
Simon Parsons,c Robert Westwooda and Peter M. Fischera,*
aCyclacel Limited, James Lindsay Place, Dundee DD1 5JJ, Scotland, UK
bUFC Limited, Synergy House, Guildhall Close, Manchester Science Park, Manchester M15 6SY, England, UK
cThe University of Edinburgh, Department of Chemistry, Joseph Black Chemistry Building, The King’s Buildings,
West Mains Road, Edinburgh EH9 3JJ, Scotland, UK
Received 22 October 2001; accepted 23 October 2001
Abstract—The cyclin-dependent kinase inhibitor (R)-2-(6-benzylamino-9-isopropyl-9H-purin-2-ylamino)butan-1-ol (roscovitine,
1a), as well as its (S)-enantiomer 1b, were synthesised. The chemical structure and absolute configuration of both enantiomers was
confirmed by X-ray crystallography. Furthermore, high enantiomeric excess (>98%) was demonstrated by chiral chromatography
of 1a and 1b, as well as NMR analysis of the diastereomeric Mosher’s ester derivatives 2a and 2b. © 2001 Elsevier Science Ltd.
All rights reserved.
reported for the racemate 1 and the enantiomers 1a and
1. Introduction
1b, i.e. 137–139, 102–104 and 118–120°C, respectively.6
In order to reconcile these questions regarding the
structure and stereochemistry of roscovitine, we decided
to establish directly both absolute configuration and to
demonstrate quantitatively the enantiomeric purity of
roscovitine and its enantiomer.
Active-site inhibitors of cyclin-dependent kinases
(CDKs) have recently gained importance as potential
therapeutics for the treatment of a range of prolifera-
tive disorders,1 particularly cancer.2 One compound
class that has yielded many CDK-selective ATP antag-
onists is the 2,6,9-trisubstituted purines.3,4 The repre-
sentative of this compound class whose biological and
pharmacological properties have been studied in most
detail is known as roscovitine.1,2,5 The synthesis of
roscovitine† 1a was first reported by Havlicek et
al.,6 who described this compound as (R)-2-(6-benzyl-
amino-9-isopropyl-9H-purin-2-ylamino)
butan-1-ol
(compound 26 in Ref. 6). There, 1a was shown as the
levorotatory enantiomer ([h]2D0=−34.6, c 0.43, CHCl3),
whereas the 2-(S)-enantiomer 1b (compound 25 in Ref.
6) was found to be dextrorotatory ([h]2D0=+35.3, c 0.57,
CHCl3). Elsewhere,7 1a was reported as having [h]2D0=
+35.1, (c 0.29, CHCl3), although the synthesis description
was incorrectly entitled as the racemic modification.
Another discrepancy arises from the melting points
2. Results and discussion
Using essentially the same synthesis methods as those
described previously,6 we found that several of our
batches (chemical purity ]95% by RP-HPLC) of
roscovitine 1a and its enantiomer 1b consistently
showed specific rotation values of opposite sign and
* Corresponding author. E-mail: pfischer@cyclacel.com
† Also referred to as (R)-roscovitine, National Cancer Institute NSC
No. 701554. Racemic material NSC No. 683246.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00471-2

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S. Wang et al. / Tetrahedron: Asymmetry 12 (2001) 2891–2894
significantly greater magnitude than previously
reported,6 i.e. [h]D20=+53 3 and [h]2D0=−53 3 (c 0.56,
CHCl3), respectively. Furthermore, we found mp
ranges of 106–108°C for all our batches, regardless of
chirality, as would be expected.
As far as biological activity is concerned, roscovitine
was reported to be two- to three-fold more potent
than its (S)-enantiomer as an inhibitor of histone H1
phosphorylation by starfish p34cdc2/cyclin B kinase,8
while the racemate had intermediate activity.6 In a
different study, the same authors confirmed this result
and showed the (R) absolute configuration of roscov-
itine through a crystal structure of roscovitine in
complex with CDK2.9 Working with recombinant
human CDK2/cyclin E kinase (histone H1 phospho-
rylation, [ATP]=0.1 mM),10 we observed a three-fold
potency difference, i.e. IC50 values of 80 20 nM and
240 20 nM for roscovitine and the (S)-enantiomer,
respectively. This result is consistent with the relevant
literature, although our IC50 value for roscovitine
against CDK2/cyclin E is approximately 10-fold lower
than that reported elsewhere, apparently using a simi-
lar assay.11
Figure 1. Molecular structure of 1a; displacement ellipsoids
enclose 50% probability surfaces. The minor disorder com-
ponent about C101 has been omitted for clarity.
In order to establish the absolute configuration of
roscovitine, we solved single-crystal structures of both
enantiomers 1a and 1b by X-ray diffraction.‡ In both
structures, which are related to one another by inver-
sion, there are two crystallographically independent
molecules, which are essentially identical except for
very minor differences in conformation. A plot of the
structure of the (R)-isomer 1a is shown in Fig. 1.
Absolute structures of light-atom organic systems can
normally be achieved by X-ray diffraction provided
there is sufficient oxygen in the system to yield a
measurable anomalous scattering signal with Cu-Ka
radiation. In the case of the present system the oxy-
gen content is only 4.5% by weight, this being com-
prised of a terminal oxygen atom exhibiting relatively
high vibrational motion. Disorder in the isopropyl
groups added further difficulties, and determination of
the absolute structures of these molecules by conven-
tional X-ray diffraction measurements with Cu-Ka
radiation was not possible. Flack has shown that the
precision of the Flack parameter (x) can be much
improved by incorporating into the refinement a few
very carefully measured data for selected enantio-sen-
sitive reflections.12 The value adopted by x is subject
to systematic errors, for example absorption, which
may drown out weak anomalous scattering effects.
We have solved this problem by performing measure-
ments at setting angles of 2q, ꢀ,  and ƒ for one
reflection and −2q, −ꢀ,  and ƒ for its Friedel oppo-
site. Under these conditions the absorption factors for
both are identical, and the quotient F2(h)/F2(−h) is
free from absorption (and extinction) errors. These
quotients were measured for each of the eight Laue
equivalents for a particular reflection, and the results
averaged. Quotients which differed significantly from
unity (7 for the (R)-isomer and 6 for the (S)-isomer)
were incorporated into the refinement in the form of
explicit restraints.13 Even for this challenging case the
Flack parameter adopted values of −0.05(5) and
0.00(6) for the (R)- and (S)-isomers, respectively.
These values are well within the limits recently
defined by Flack for strong enantio-distinguishing
power.14 Full details of this method will be published
separately.
We also attempted to measure the enantiomeric
purity of our preparations of 1a and 1b. Clean ana-
lytical separation of the enantiomers was achieved
using chiral HPLC and an e.e. value of 98.3 1.8%
was derived for roscovitine 1a. Conversion of the
enantiomers into diastereomeric esters using Mosher’s
method15 was also performed. Derivatives 2a and 2b
were prepared with the aid of highly enantiomerically
pure (R)-(−)-3,3,3-trifluoro-2-methoxy-2-phenylpropi-
onyl chloride in the usual manner. Examination of
1
the H NMR spectra of 2 showed near-baseline sepa-
ration of the multiplet signals arising from the
methoxy protons of 2a and 2b, respectively. It was
therefore possible to integrate these and to estimate
e.e. values of ]98% for both 2a and 2b, consistent
with the chiral HPLC data. Separation of the
diastereomeric CF3 signals in the 19F NMR spectra of
2 was also observed but resolution was not sufficient
for quantitation purposes.
‡ Crystallographic data (excluding structure factors) for both struc-
tures reported in this paper have been deposited with the Cam-
bridge Crystallographic Data Centre as deposition Nos. CCDC
157779 and 157780. Copies of the data can be obtained, free of
charge, on application to the CCDC, 12 Union Road, Cambridge
CB2 1EZ UK (fax: +44 (1223) 336 033; e-mail: deposit@
ccdc.cam.ac.uk).

S. Wang et al. / Tetrahedron: Asymmetry 12 (2001) 2891–2894
2893
In conclusion, our data confirm conclusively that
roscovitine 1a does indeed possess the R absolute
configuration but is the dextrorotatory enantiomer,
whereas the S enantiomer 1b is levorotatory. Further-
more, we demonstrate high enantiomeric purity for
our preparations of 1a and 1b.
(S)-2-(6-Benzylamino-9-isopropyl-9H-purin-2-ylamino)
butan-1-ol 1b. This was prepared in the same way as
the (R)-enantiomer 1a from 6-benzylamino-2-chloro-9-
isopropyl-9H-purine with the exception that (S)-(+)-2-
amino-1-butanol ([h]2D0=+10.0 (neat), 96% e.e./GLC)
was used: colourless crystalline solid of 1b. Mp 108–
109°C (AcOEt/hexane), [h]2D0=−56.3 (c 0.56, CHCl3).
RP-HPLC: tR=17.3 min (0–60% B in A over 20 min),
tR=18.8 min (23–33% B in A over 20 min), purity
]99.5% (by integration at u=214 nm); admixtures of
1a and 1b co-eluted. Spectroscopically 1b was indistin-
guishable from 1a.
3. Experimental
3.1. General
Column chromatography:16 Merck silica gel 60, 230–
400 mesh. Optical rotation: Polaar 2001 (Optical
Activity Ltd.) polarimeter. RP-HPLC: Vydac 201HS54
column, 4.6×250 mm, eluant A: 0.1% CF3COOH in
H2O, B: 0.1% CF3COOH in MeCN; flow rate 1 mL/
min, 25°C. Mp: Reichert hot-stage microscope melting
point apparatus; uncorrected. NMR spectra: Bruker
DMX 500 NMR spectrometer; l values in ppm rel. to
SiMe3 (1H NMR) and CFCl3 (19F NMR; hexa-
fluorobenzene was used as a secondary reference
(−163 ppm)). Coupling constants J in Hz.
3.3. Separation of enantiomers by chiral HPLC
A Chiralpak AD column (4.6×250 mm, 10 mm parti-
cles;
c
AD00CE-AK048, from Daicel Chemical
Industries, Tokyo, Japan) was used. Isocratic elution
with hexane/propan-2-ol (3:1, v/v) at 1 mL/min (20°C)
was performed and quantitation was achieved by inte-
gration of UV-absorption (u=210 nm) peaks. Samples
were prepared at 1 mg/mL in MeOH and 10 mL
aliquots were injected. tR=12.6 min 1a, 19.0 min 1b.
Limits of detection and quantitation were determined
using admixture solutions of 1a and 1b in various
proportions. The lower limit of quantitation was
found to be 0.1%. For six different batches of 1a
e.e.=98.3 1.8% (mean standard deviation) was thus
determined.
3.2. Synthesis of roscovitine enantiomers
(R)-2-(6-Benzylamino-9-isopropyl-9H-purin-2-ylamino)
butan-1-ol 1a. A mixture of 6-benzylamino-2-chloro-9-
isopropyl-9H-purine, prepared as described,6 (30.2 g,
0.1 mol) and excess (R)-(−)-2-amino-1-butanol (60
mL; [h]2D0=−9.78 (neat), 96% e.e./GLC) was heated at
160°C for 5 h in three sealed tubes with stirring. After
cooling to room temperature, the combined reaction
mixtures were diluted with CHCl3 (450 mL), extracted
with H2O (3×210 mL), dried on MgSO4, filtered and
evaporated. The residue was purified by flash chro-
matography (120 g silica gel, 0–4% MeOH/CH2Cl2)
and recrystallised from AcOEt/hexane to afford solid
pale-green crude product (27.2 g, 77%). This material
was re-chromatographed (120 g silica gel, AcOEt)
twice and again recrystallised from AcOEt/hexane:
colourless crystalline solid of 1a (21.0 g, 59%). Mp
106–108°C. [h]D20=+56.3 (c 0.56, CHCl3). 1H NMR
(500 MHz, CDCl3): 1.01 (t, 3J=7.5, 3H, CH2CH3);
1.51 (dd, 3J=7.5, 6H, CH2(CH3)2); 1.55 (m, 1H,
CH2CH3); 1.62 (m, 1H, CH2CH3); 3.61 (dd, 2J=11,
3J=8, 1H, CH2OH); 3.80 (dd, 2J=11, 3J=3, 1H,
CH2OH); 3.88 (m, 3J=8, 6, 3, 1H, CHNH); 4.58
(hept., 3J=7.5, 1H, CH(CH3)2); 4.75 (bm, 2H,
3.4. Crystal structure determinations
Single crystals of 1a and 1b were grown from AcOEt/
iPr2O. 1a C19H26N6O, M=354.46, orthorhombic, a=
,
A,
11.4105(3),
b=15.4752(4),
c=22.4839(7)
3
,
U=3970.20(19) A , T=220 K, space group P212121,
Z=8, ((Cu-Ka)=0.618 mm−1. Refinement was per-
formed against F with 5378 data with F>4|(F). The
final R-factor was 3.72%. 1b C19H26N6O, M=354.46,
orthorhombic, a=11.4045(11), b=15.4797(18), c=
3
,
,
22.489(3) A, U=3970.1(8) A , T=220 K, space group
P212121, Z=8, ((Cu-Ka)=0.618 mm−1. Refinement
was performed against F with 6010 data with F>
4|(F). The final R-factor was 4.19%.
3.5. Synthesis and analysis of Mosher’s ester
derivatives
(R,R)-2a
and
(R,S)-3,3,3-Trifluoro-2-methoxy-2-
3
CH2Ph); 4.87 (bd, J=6, 1H, CHNH); 5.09 (bs, 1H,
phenylpropionic acid 2-(6-benzylamino-9-isopropyl-
9H-purin-2-ylamino)butyl ester 2b. Enantiomer 1a or
1b (50 mg, 0.14 mmol) was dissolved in CDCl3 (1
mL). To the stirring solution was added (R)-(−)-3,3,3-
trifluoro-2-methoxy-2-phenylpropionyl chloride (54 mL,
73 mg, 0.29 mmol; ]99% e.e./GLC). The reactions
were complete after 5 min (TLC, 95:5 CH2Cl2/MeOH
OH); 6.09 (bs, 1H, NH); 7.23–7.36 (m, 5H, Ph); 7.44
(s, 1H, purine H(8)). 13C NMR (125 MHz, CDCl3):
10.91 (CH2CH3); 22.51, 22.58 (CH(CH3)2); 25.00
(CH2CH3); 44.42 (CH2Ph); 46.42 ((CH3)2CH2); 56.35
(CHNH); 68.51 (CH2OH); 114.68 (purine C(5));
127.34 (Ar-CH, para); 127.71, 128.58 (Ar-CH, ortho/
meta); 134.58 (purine CH(8)); 138.71 (Ar-C); ꢀ152
(purine C(4)); 154.81 (purine C(6)); 159.96 (purine
C(2)); RP-HPLC: tR=17.3 min (0–60% B in A over
20 min), tR=18.8 min (23–33% B in A over 20 min),
purity ]99.5% (by integration at u=214 nm). Anal.
calcd for C19H26N6O (354.45): C, 64.38; H, 7.39; N,
23.71; found: C, 64.5; H, 7.3; N, 23.9.
1
and H NMR). The reaction mixtures were evaporated
and the residues were purified by flash chromatogra-
phy (3.5 g silica gel, 0–2% MeOH in CH2Cl2). The
diastereomeric esters 2a and 2b were obtained as clear
gums after evaporation and drying in vacuo (ca. 80%).
1H NMR (500 MHz, CDCl3) of admixtures or pure 2a
and 2b: inter alii 3.50 (m, J=0.64, OCH3, (R,S)); 3.52

2894
S. Wang et al. / Tetrahedron: Asymmetry 12 (2001) 2891–2894
(m, J=0.85, OCH3, (R,R)). 19F NMR (282.4 MHz,
CDCl3): −68.12 (s, (R,S)), −68.20 (s, (R,R)).
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Acknowledgements
We thank Simon Olley and Craig McNair (Onyx Scien-
tific Limited, Sunderland, England) for the chiral
HPLC work and Dr John A. Parkinson (Department of
Chemistry, University of Edinburgh, Scotland) for
NMR spectroscopy. We also thank Dr. R. I. Cooper
(University of Oxford) for modifications to the pro-
gram CRYSTALS to enable the crystallographic
restraints described here to be applied.
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