Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: Key intermediate for Src-SH2 inhibitor

Article abstract of DOI:10.1016/S0960-894X(02)00256-1

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.

Full text of DOI:10.1016/S0960-894X(02)00256-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1735–1738
Efficient Chemoenzymatic Synthesis of (S)- and
(R)-5-(1-Aminoethyl)-2-(cyclohexylmethoxy)benzamide:
Key Intermediate for Src-SH2 Inhibitor
Ahmed Kamal* and Mahendra Sandbhor
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received 7 March 2002; accepted 16 April 2002
Abstract—A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a
key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity
(>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia
lipase offered high degree of enantioselectivity with spontaneity. # 2002 Elsevier Science Ltd. All rights reserved.
Src-homology-2 (SH2) domain and their associated
catalytic or non-catalytic protein constitute critical sig-
nal transduction target for drug discovery, which is both
challenging and promising with respect to the design of
novel inhibitors. SH2 domain are found in the regula-
tion of number of cellular processes, including growth,
mitogenesis, motility, metabolism, immune response
and gene transcription.1 Of particular interest are inhi-
are attained in the binding of these peptides with SH2
domain. First, the NH of the amide attached to phos-
phophenyl group is a hydrogen bond donor to His 58
(CO) of the Src-SH2 domain, thus mimicking the inter-
action observed with P+112 NH in the 11-mer.13 Sec-
ond, and more interestingly, the carboxamide
substituted on phenyl in the linker template displaces
water molecules found in the 11-mer X ray structure
and binds directly to Lys 60 (NH) of Src-SH2 domain.
bitors of the SH2 domain of the tyrosine kinase pp60c-Src
,
which has been implicated as a potential target2 for the
therapeutic intervention for both osteoporosis3 and
breast cancer.4 There has been interest to develop com-
pletely non-peptidic Src-SH2 inhibitors based on phos-
photyrosine (pTyr) portion of tetrapeptide 1 (Fig. 1), a
high affinity ligand for the Src-family SH2 domain.
Recently a non-peptidic, monocharged compound has
been designed which displays good binding affinity for
the p561ck SH2 domain and good cell permeation
properties.5 There are some reports6ꢀ8 that have exam-
ined pTyr replacement attached to a novel non-peptidic
1-aminoethyl substituted benzamide for the Glu-Glu-
Ile-NH2 within ‘tail’ portion of 1. This modified com-
pound 2 exhibits improved binding affinity over 1,
whereas compound 3, which possesses same ‘tail’ but
attached to a 4-carboxyphenyl phosphate ‘head’ group,
shows comparable binding affinity as 1.9ꢀ11 The crys-
tallographic study8 shows that two key polar contacts
We have been interested in the development of non-
peptidic Src-SH2 inhibitors and to investigate an effi-
cient chemoenzymatic enantioselective methodology
employing lipases. In this context, the starting materials
for the key intermediate 5 of non-peptidic Src SH2
inhibitors of type 2–4 that contains acetophenone moi-
ety provides the required secondary alcohol upon
reduction, which was resolved by lipase-mediated acy-
lation in the same pot. The synthesis of non-peptidic
Src-SH2 inhibitor, reported in the literature for this
compound has either employed racemic amine 5 or one
of the enantiomerically enriched S amine 5b.
In continuation of our efforts towards the synthesis of
biologically important chiral compounds employing
enzymes,14,15 it is considered of interest to enzymatically
resolve the precursors of this amine 5, which would
allow the preparation of both the R and S enantiomers.
The chiral synthesis reported in the literature for this key
intermediate involves reduction of methyl 5-acetyl-2-
(cyclohexylmethoxy)benzoate 6 using (+)DIP-chloride
*Corresponding author. Tel.: +91-40-719-3157; fax: +91-40-716-
0512; e-mail: ahmedkamal@iict.ap.nic.in
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00256-1

1736
A. Kamal, M. Sandbhor / Bioorg. Med. Chem. Lett. 12 (2002) 1735–1738
at ꢀ13 ꢁC for 2.5days to afford the corresponding
alcohol in 94% ee, which leads to the required amine 5b
in 88% ee. Whereas, another protocol employs the
chiral 1-(4-methoxy-phenyl)ethylamine as the precursor,
which yielded the required amine 5b in 94% ee.9 How-
ever, no efforts have been made in the literature to pre-
pare both the R and S forms of the amine 5, which
could be of significance to unravel the correlation
between stereoselectivity and biological activity of these
inhibitors.
wheat germ source were inactive towards this one-pot
acetylation. Further there was no significant conversion
employing lipase from Candida antarctica, Candida
cylindracea, Candida rugosa and Mucor miehei (lipo-
zyme). Lipase from Amano-PS and Ps. fluorescence has
shown effective conversion in 42 h with high enantio-
selectivity in acetate 8. Amongst all, the immobilized
lipase PS-C ‘Amano’ II afforded good conversion and
high degree of selectivity in shorter duration (Table 1).
In a typical representative procedure for lipase-medi-
ated acylation, to a solution of the 6 (1 mmol) in 10 mL
of hexane was added moist neutral alumina (1.0 g) and
NaBH4 (2 mmol). The resulting reaction mixture was
stirred at 40 ꢁC for 3 h and monitored for the comple-
tion of the reduction by TLC. At the end of the reaction
was added lipase PS-C ‘Amano’ II (1 equiv w/w) and
isopropenyl acetate (0.65mL). The reaction was stirred
at room temperature for 5–6 h till the reaction reached
to 50% conversion. The reaction was filtered through
Celite, diluted with EtOAc and washed with water. The
organic layer was dried over anhydrous sodium sulfate
and concentrated under reduced pressure to give an oily
residue, which was purified by silica gel column chro-
matography to obtain enantiomerically pure acetate and
alcohol.17 Products, thus obtained were analyzed by
chiral HPLC and compared with corresponding racemic
products.18
In an attempt to exploit the synthesis of the key inter-
mediate for SH2 inhibitor, herein we report for the first
time, a new chemoenzyamtic method that produces the
chiral amine 5 with high degree of selectivity. Methyl 5-
acetyl-2-(cyclohexylmethoxy)benzoate 6 was reduced
using sodium borohydride and moist neutral alumina in
hexane followed by lipase-mediated resolution in the
same pot to afford the corresponding alcohol 7 and
acetate 8 in high enantiomeric purity (Scheme 1).
One mmol of 6 was reduced by sodium borohydride
with moist neutral alumina16 in hexane at 40 ꢁC and
subjected for lipase-mediated resolution process (1 equiv
w/w) at room temparature. It was observed that immo-
bilized lipase; PS-C ‘Amano’ II and PS-D ‘Amano’ I
have shown high degree of selectivity both in alcohol 7
and acetate 8. Lipase from porcine pancrease (PPL) and
Figure 1.
Scheme 1. Reagents and conditions: (a) NaBH4, moist Al2O3 (neutral), hexane, lipase PS-C ‘Amano’ II, isopropenyl acetate, 50% (7), 47% (8).

A. Kamal, M. Sandbhor / Bioorg. Med. Chem. Lett. 12 (2002) 1735–1738
1737
Scheme 2. Reagents and conditions: (a) K2CO3, MeOH, 98%; (b) HOBt, EDC, NMM, NH4OH, THF, 91%; (c) DPPA, DBU, toluene, 71%;
(d) 10% Pd/C, H2, EtOAc, 88%; (e) CH3COCl, Et3N, CH2Cl2, 99%.
Table 1. In-situ transesterification employing different lipases
Acknowledgements
Lipase
Time (h)a
Conversion (%)b
ee (%)c
We are thankful to the Department of Science and
Technology, New Delhi for the financial support for
Grants-in-Aid project under SERC (No. SP/S1/G-47/
99). Generous gift of P. cepacia lipases (PS-C ‘Amano’
II and PS-D ‘Amano’ I) by Amano Pharmaceutical
Company (Japan) are gratefully acknowledged. One of
the authors (M.S.) is thankful to CSIR (New Delhi) for
the award of a Junior Research Fellowship.
7
8
PS ‘Amano’
42
6
12
42
46
50
50
40
84
>99
>99
>99
>99
PS-C ‘Amano’ II
PS-D ‘Amano’ I
Ps. fluorescence
>99
>99
65
aTime taken for transesterification at which the ratio of 7/8 remains
constant.
ee7
bCalculated by; % conversion= ee þee  100.
7
8
cDetermined by chiral HPLC.
References and Notes
1. Lawrence, D. S.; Niu, J. Pharmacol. Ther. 1998, 77, 81.
2. Thomas, S. M.; Brugge, J. S. Annu. Rev. Cell Dev. Biol.
1997, 13, 513.
3. Brown, M. D.; Cooper, J. A. Biochim. Biophys. Acta 1996,
199, 1287.
Chiral alcohol 7 and acetate 8 were further transformed
to the highly enantiopure amine 5a and 5b by employing
the modified procedures of Luke and Holt (Scheme 2).9
The alcohol 7 and acetate 8 were hydrolyzed indepen-
dently by potassium carbonate in methanol to their
corresponding benzoic acids 9a and 9b, which were fur-
ther converted to their carboxamides 10a and 10b. Sub-
stitution of secondary hydroxy group by an azide was
carried out employing diphenyl phosphorylazide, that
usually involves the change of configuration at the
chiral center, but in this case interestingly no racemiza-
tion was observed. Finally, the azide was reduced to the
desired (R) and (S) 5-(1-aminoethyl)-2-(cyclohexyl-
methoxy)benzamide (5) which was isolated in high
enantiomeric excess (5a ee >99%; 5b ee >99%).19 The
enantioselectivity was determined by chiral HPLC
studies through their acetyl derivative (12).20
4. Soriano, P.; Montgomery, C.; Geske, R.; Bradley, A. Cell
1991, 64, 693.
5. Proudfoot, J. R.; Rajashehar, B.; Cardozo, M.; Gilmore,
A. T.; Glynn, S.; Hickey, R. E.; Jakes, S.; Kabcenell, A.; Kir-
rane, M. T.; Tibolla, A. T.; Lukas, S.; Usha, P. R.; Sharma,
R.; Yazdanian, M.; Moss, N. J. Med. Chem. 2001, 44, 2421.
6. Buchanan, J. L.; Bohacek, R. S.; Luke, G. P.; Hatada, M.;
Lu, X.; Dalgarno, D. C.; Narula, S. S.; Yuan, R.; Holt, D. A.
Bioorg. Med. Chem. Lett. 1999, 9, 2353.
7. Buchanan, J. L.; Vu, C. B.; Merry, T. J.; Corpuz, E. G.;
Pradeepan, S. G.; Mani, U. N.; Yang, M.; Plake, H. R.; Var-
khedkar, V. M.; Lynch, B. A.; MacNeil, I. A.; Tiong, C. L.;
Holt, D. A. Bioorg. Med. Chem. Lett. 1999, 9, 2359.
8. Lunney, E. A.; Para, K. S.; Rubin, J. R.; Humblet, C.;
Fergus, J. H.; Marks, J. S.; Sawyer, T. K. J. Am. Chem. Soc.
1997, 119, 12471.
9. Luke, P. G.; Holt, A. D. Tetrahedron: Asymmetry 1999, 10,
4393.
10. Sawyer, T. K. Biopolymers 1998, 47, 243.
11. Bohacek, R. S.; Dalgarno, D. C.; Hatada, M.; Jacobsen,
V. A.; Lynch, B. A.; Macek, K. J.; Merry, T.; Metcalf, C. A.,
III; Narula, S. S.; Sawyer, T. K.; Shakespeare, W. C.; Violette,
S. M.; Weigele, M. J. Med. Chem. 2001, 44, 660.
12. Nomenclature for binding sites designates P for pTyr
site. Residues N-terminal to P are P-1, P-2 and so on;
residue C-terminal to P are P+1, P+2, and so on.
In summary, one-pot protocol was developed for the
NaBH4/Al2O3 reduction accompanied by in-situ lipase-
mediated transesterification, for the synthesis of both R
and S forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)-
benzamide, an important intermediate required for the
preparation of structurally modified non-peptidic Src-
SH2 inhibitors. Both the enantiomers were synthesized
in high optical purity for the first time and could assist
researchers to design and synthesize novel analogues in
the desired stereochemical forms.

1738
A. Kamal, M. Sandbhor/ Bioorg. Med. Chem. Lett. 12 (2002) 1735–1738
13. Waksman, G.; Sheoelson, S. E.; Pant, N.; Cowburn, D.;
Kuriyan, J. Cell 1993, 72, 779.
19. Spectral data for compound 5a (white solid): IR (KBr)
1
1653, 2923, 3184, 3492 cmꢀ1; H NMR (200 MHz, CDCl3) d
14. Kamal, A.; Ramana, K. V.; Rao, M. V. J. Org. Chem.
2001, 65, 997.
15. Kamal, A.; Khanna, G. B. R. Tetrahedron: Asymmetry
2001, 12, 405.
1.0–1.4 (5H, m), 1.5 (3H, d, J=6.59 Hz), 1.7–2.0 (6H, m), 3.9
(2H, d, J=5.86 Hz), 4.1 (1H, q, J=6.59 Hz), 5.8 (1H, br s), 6.9
(1H, d, J=8.80 Hz), 7.4 (1H, dd, J=8.79, 2.20 Hz), 7.8 (1H, br
s), 8.1 (1H, d, J=2.20 Hz); 13C NMR (200 MHz, CDCl3) d
167.36, 156.29, 140.18, 130.38, 129.64, 120.56, 112.49, 74.69,
50.44, 37.49, 29.96, 26.24, 25.57, 25.53; MS (FAB) m/z 277
(M++1); HRMS data: calcd for C16H25N2O2 277.191603,
16. Neutral alumina (S. d. fine chemicals, 10.0 g) was dried in
an oven at 500 ꢁC for 6 h; distilled water (1.1 mL) was added
portionwise while shaking at every min, to afford free-flowing
moist neutral alumina.
25
found 277.192082; mp 123.4 ꢁC; ½aꢂD +23.7 (c 0.8, CHCl3).
25
17. Spectral data for compound 7: ½aꢂD ꢀ30.77 (c 1.0,
Spectral data for compound 5b (white solid): IR (KBr) 1653,
;
2923, 3169, 3415cm ꢀ1 1H NMR (200 MHz, CDCl3) d 1.0–
CHCl3); IR (KBr) 3424, 2928, 1712 cmꢀ1
;
1H NMR
(200 MHz, CDCl3) d 1.1–1.4 (5H, m), 1.5 (3H, d, J=6.11 Hz),
1.6–1.9 (6H, m), 3.8 (2H, d, J=4.90 Hz), 3.9 (3H, s), 4.8 (1H,
q, J=6.11 Hz), 6.9 (1H, d, J=8.55 Hz), 7.4 (1H, dd, J=8.55,
2.45Hz), 7.7 (1H, d, J=2.45Hz), MS (EI) m/z 292 (M+).
1.4 (5H, m), 1.5 (3H, d, J=6.59 Hz), 1.7–2.0 (6H, m), 3.9
(2H, d, J=5.86 Hz), 4.1 (1H, q, J=6.59 Hz), 5.8 (1H, br s),
6.9 (1H, d, J=8.79 Hz), 7.4 (1H, dd, J=8.79, 2.20 Hz), 7.8
(1H, br s), 8.1 (1H, d, J=2.20 Hz); 13C NMR (200 MHz,
CDCl3) d 167.33, 156.30, 139.79, 130.44, 129.63, 120.54,
112.48, 74.66, 50.39, 37.43, 29.90, 26.19, 25.52, 25.14; MS
(FAB) m/z 277 (M++1); HRMS data: calcd for C16H25N2O2
25
Spectral data for compound 8: ½aꢂD +85.45 (c 1.1, CHCl3); IR
(KBr) 2928, 1728 cmꢀ1; 1H NMR (200 MHz, CDCl3) d 1.1–1.3
(5H, m), 1.5 (3H, d, J=7.33 Hz), 1.7–1.9 (6H, m), 2.0 (3H, s),
3.8 (2H, d, J=6.11 Hz), 3.9 (3H, s), 5.8 (1H, q, J=7.33 Hz),
6.9 (1H, d, J=8.56 Hz), 7.4 (1H, dd, J=8.56, 2.45 Hz), 7.8
(1H, d, J=2.45Hz); MS (EI) m/z 334 (M+).
25
277.191603, found 277.191445; mp 123.8 ꢁC; ½aꢂD ꢀ23.3 (c 1.2,
CHCl3).
20. Enantioselectivity of the amine 5a and 5b was determined
through their acetates 12a and 12b by chiral HPLC (Chiracel,
OJ-H column, Daicel) employing hexane/isopropanol/diethyl-
amine=90/10/0.1 (v/v/v) as mobile phase, 0.5mL/min flow
and monitored at 254 nm wavelength.
18. Enantioselectivity of 7 and 8 was determined by chiral
HPLC (Chiracel OD column, Daicel) employing hexane/iso-
propanol=90/10 (v/v) as mobile phase, 0.5mL/min flow and
monitored at 254 nm wavelength.