Synthesis of a metabolite of an anti-angiogenic lead candidate based on a d-glucosamine motif

Article abstract of DOI:10.1016/j.tetlet.2008.06.009

A rapid synthetic access to ACL 21269 was established in 12 steps starting from thiogylcoside 9 utilizing synthons 8 and 6 to introduce the pharmacophores at positions 1 and 2. The functional groups decorating the glucosamine scaffold were introduced in a particular order and common protecting groups were employed to establish a robust synthetic process.

Full text of DOI:10.1016/j.tetlet.2008.06.009

Tetrahedron Letters 49 (2008) 4854–4856
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a metabolite of an anti-angiogenic lead candidate
based on a D-glucosamine motif
*
Latika Singh, Ann Lam, Rajaratnam Premraj, Joachim Seifert
ALCHEMIA Ltd, 3 HiTech Court, Brisbane Technology Park, Eight Mile Plains, Brisbane, QLD 4113, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A rapid synthetic access to ACL 21269 was established in 12 steps starting from thiogylcoside 9 utilizing
synthons 8 and 6 to introduce the pharmacophores at positions 1 and 2. The functional groups decorating
the glucosamine scaffold were introduced in a particular order and common protecting groups were
employed to establish a robust synthetic process.
Received 8 April 2008
Revised 28 May 2008
Accepted 3 June 2008
Available online 6 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Anti-angiogenesis
Metabolite
Glucosamine scaffold
VASTTM
Inhibition of tissue neovascularization has presented a major
therapeutic advantage in the treatment of cancer and age-related
macular degeneration (AMD)1 where several anti-angiogenic drugs
are currently approved. The concept of presenting pharmacophoric
groups around carbohydrate motifs to bind to pharmacologically
important receptors has been exploited in the past and was first
published by Hirschmann et al. and other groups.2 Alchemia has
developed a VASTTM (Versatile Assembly on Stable Templates) drug
discovery platform,3a,b a technology based on carbohydrate scaf-
folds, which are used in the production of focused libraries. Using
the VASTTM technology, lead candidates have been produced, which
appear through their anti-angiogenic activity to have application in
the therapeutic areas oncology, AMD and diabetic retinopathy.
During the non-clinical characterization of a VASTTM anti-cancer
lead candidate, it was demonstrated that this compound was
microsomally metabolized into a novel intermediate, which exhib-
ited a long half-life and novel structural elements. Its structure is
shown in Scheme 1 and the compound is further referred to as
ACL 21269. To further elucidate the pharmacokinetic properties
of this compound, a rapid synthetic access was developed utilizing
readily available thioglycoside 94 (Scheme 4) as starting material.
We devised a synthetic strategy (Scheme 4), which involved 12
Scheme 1. ACL 21269 was synthesized using p-thiocresyl glycoside 9 and synthons
8 and 6 for introducing the pharmacophores at positions 1 and 2.
methyl moiety (2NM) was introduced first, and the position 6
was O-methylated only after protecting the 4-OH-group. The acid
labile m-hydroxyphenethyl moiety 8 was introduced via glycosyl-
ation to the protected thioglycoside 16. The benzoyl protection in
the aglycon 8 further enhanced the stability of the phenethyl gly-
coside 17
group, the
under standard coupling conditions utilizing
a
, towards acidic cleavage. After reduction of the 2-azido
-aminobutyrate (‘GABA’) side chain was introduced
-azidobutyrate 6 as
c
protecting group manipulations around a
D-glucosamine motif.
c
Common protecting groups were applied to facilitate scalability
of the individual steps. It was important to decorate the scaffold
with the pharmacophoric groups in a certain order and to use
properly adjusted protecting groups. Therefore, the 2-naphthyl-
the synthon. The overall deprotection was achieved in two steps
to produce the target molecule in high yields.
The synthesis of the GABA-synthon 6 was executed in five steps,
starting from N-DTPM-protected
c
-aminoalcohol 15a–c and is
shown in Scheme 2. Silylation of alcohol 1 followed by removal
of the amino protection and ensuing diazo transfer reaction6 led
to c-azido-butanol 4. Subsequent removal of the silyl ether in 4
* Corresponding author. Tel.: +61 7 3340 0244; fax: +61 7 3340 0222.
E-mail address: jseifert@alchemia.com.au (J. Seifert).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.06.009

L. Singh et al. / Tetrahedron Letters 49 (2008) 4854–4856
4855
aqueous work-up and short silica gel column chromatography to
furnish compound 8 as a pale yellow syrup.
Having prepared the side chain synthons 6 and 8, the synthesis
of thioglycoside 16 was next pursued (Scheme 4). The first six steps
of the synthesis (steps a–f) were performed under standard condi-
tions and did not involve any chromatography, since most of the
intermediates were obtained in high purities via precipitation. It
is noteworthy that due to limited steric access to the 4-OH-group
in 13, the benzoylation to compound 14 required heating (60 °C),
the addition of DMAP and extended reaction time. The introduc-
tion of the methyl ether on 15 (step g) was initially attempted
under basic conditions using tBuOK as base9a and varying the
solvents and temperatures (data not shown). In all cases, we
observed migration of the benzoyl group to the 6 position (16a,
Scheme 5) concomitant with varied amounts of 4-O-methylation
(16b, Scheme 5). The product 16 and side products 16a and 16b
appeared at well separated retention times on the HPLC and were
also isolated and the structures verified via NMR spectroscopy.10a–c
Use of milder conditions such as MeOTf/2,6-DTBP9b gave sluggish
reactions with multiple side products.
Scheme 2. Synthesis of
c-azido butyric acid 6. Reagents and conditions: (a)
TBDPSCl, imidazole, DMF, (89%); (b) 5% N2H4 in MeOH;5b (c) TfN3, DMAP, MeCN,
CH2Cl2,6 (66%, 2 steps); (d) TBAF, HOAc, THF (77%); (e) NaOCl, NaClO2, TEMPO, pH
7.8, H2O, CH3CN7 (88%).
followed by TEMPO-mediated oxidation of the hydroxy group with
NaOCl/NaClO2 in a phosphate buffer7 facilitated the carboxylic acid
6 as a brown viscous liquid.
The synthesis of the benzoyl protected m-hydroxyphenethyl
aglycon 8 was achieved in two steps, starting from unprotected
m-hydroxyphenethyl alcohol 7 (Scheme 3). First, alcohol 78a was
transformed into the cesium phenolate 7a. Subsequent reaction
of crude 7a with benzoyl chloride under vigorous stirring produced
aglycon 8 in good yield. Finally, purification was accomplished by
We next shifted our focus to perform the transformation under
acidic conditions. The obvious choice for this purpose would have
been the use of diazomethane.11a,b However, due to its hazardous
nature, we replaced it with TMSCHN2.11c Stirring of 15 with
TMSCHN2 over silica gel11d in CH2Cl2 also resulted in a sluggish
reaction. Therefore, we next applied stronger acids to effect this
11e
conversion. Methylation via aqueous HBF4 and TMSCHN2
gave
ꢀ70% of the product 16 and trehalose 16d10d (Scheme 5) as the ma-
jor byproduct. Alternatively, BF3ꢁOEt2 as Lewis acid11f and
TMSCHN2 facilitated the conversion in a clean fashion but stopped
at ꢀ50% conversion with 16c (Scheme 5) as the only side product.
We modified the conditions such that, following methylation, the
Scheme 3. Synthesis of aglycon 8. Reagents and conditions: (a) CsOH, H2O; (b) BzCl,
CH2Cl2 (73%, 2 steps).
Scheme 4. Synthesis of ACL 21269 starting from thioglycoside 9. Reagents and conditions: (a) (CH3)2C(OMe)2, CSA, DMF, (72%); (b) 2-NMBr, DMF, NaH, 0 °C (98%); (c) TFA,
CH2Cl2, H2O (95%); (d) TBDPSCl, imidazole, DMF, 60 °C, 2 h; (e) BzCl, pyridine, DMAP, 60 °C, 18 h (92%, 2 steps); (f) TBAF, HOAc, THF, 18 h (91%); (g) (i) TMSCHN2, cat. BF3ꢁOEt2,
CH2Cl2, 0 °C; (ii) TBAF, HOAc, THF (48%); (h) 8, MeOTf, diethyl ether, MS 4 Å, 5 °C to rt (31% of 17a, 19% of 17b); (i) Zn, NH4Claq, DMF, MeOH, (98% crude); (j) N3–(CH2)3–CO2H
(6), DCC, DIPEA, CH2Cl2 (85%); (k) 1 M LiOH, H2O2, THF, 18 h (87%); (l) Zn, NH4Claq, DMF, MeOH, 2 h (91%).

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L. Singh et al. / Tetrahedron Letters 49 (2008) 4854–4856
Halliday, J.; Tometzki, G.; Zuegg, J.; Meutermanns, W. Drug Discovery Today
2003, 8, 701.
4. Compound 1 is accessible from 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl acetate4d in
3 steps: (a) p-Cres-SH, SnCl4, CH2Cl2 ; (b)
4e
(H2NCH2)2, n-BuOH, 100 °C;4g (c) Tf–N3,4f CuSO4, Et3N, CH2Cl2, MeOH (63%, 3
steps); (d) Lemieux, R. U.; Takeda, T.; Chung, B. Y. A.C.S. Symp. Ser. 1976, 39, pp
90–115; (e) Loenn, H. Carbohydr. Res. 1985, 139, 105; (f) Alper, P. B.; Hung, S.-C.;
Wong, C.-H. Tetrahedron Lett. 1996, 37, 6029; (g) Kanie, O.; Crawley, S. C.; Palcic,
M. M.; Hindsgaul, O. Carbohydr. Res. 1993, 243, 139.
5. (a) Singh, L.; Seifert, J. Tetrahedron Lett. 2001, 42, 3133; (b) Dekany, G.;
Bornaghi, L.; Papageorgiou, J.; Taylor, S. Tetrahedron Lett. 2001, 42, 3129; (c)
compound 1 is accessible by reaction of 4-amino-1-butanol with a solution of
DTPM-NMe2 in MeOH.5b
6. Vasella, A.; Witzig, Ch.; Chiara, J.-L.; Martin-Lomas, M. Helv. Chim. Acta 1991,
74, 2073.
Scheme 5. Observed side products during the methylation of 15.
7. Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D. M.; Grabowski, E. J. J.; Reider, P. J.
J. Org. Chem. 1999, 64, 2564.
8. (a) 3-Hydroxyphenethyl alcohol was purchased from Sigma Aldrich.; (b) Fmoc-
4-aminobutyric acid (Fmoc-GABA) was purchased from Neo MPS (Strasbourg,
crude reaction mixture was treated with TBAF. Subsequent chro-
matography furnished product 16 in 48% yield with almost com-
plete recovery of unreacted starting material 15.
For the coupling of thioglycoside 16 with aglycon 8 (step h), we
evaluated NIS/TMSOTf12a and MeOTf12b,c as promoters in combina-
tion with varying solvents and temperatures. These experiments
showed that the b-glycoside 17b13a was the preferred product
France).; (c) Boc-
Biochem.
c-Aminobutyric acid (Boc-GABA) was purchased from Nova
9. (a) Greene, T. W.; Wuts, P. G. M. Protecting Groups in Organic Synthesis, 3rd ed.;
John Wiley & Sons, 1999. pp 23–27; (b) Marshall, J. A.; Xie, S. J. Org. Chem. 1995,
60, 7230.
10. Selected NMR data of intermediates; 1H NMR spectra were recorded at
400 MHz, 13C NMR at 100 MHz; the solvent is mentioned in brackets after the
compound number, (a) Compound 16 (CDCl3); 1H NMR: d = 5.17 (dd, 1H,
J3,4ꢀJ4,5 = 9.6 Hz, H-4), confirming a benzoyl ester at position 4.(b) Compound
16a (CDCl3); 1H NMR: d = 4.74 (dd, 1H, J5,6a = 3.5 Hz, Jgem = 11.9 Hz, H6a), 4.56
(dd, 1H, J5,6b = 2.2 Hz, Jgem = 11.9 Hz, H6b), 4.41 (d, 1H, J1,2 = 10.1 Hz, H-1b), 3.54
(m, 1H, H-5), 3.49 (dd, 1H, not assigned), 3.43 (dd, 1H, not assigned), 3.27 (dd,
1H, not assigned).(c) Compound 16b (CDCl3); 1H NMR: d = 4.74 (dd, 1H,
J5,6a = 2.2 Hz, Jgem = 11.9 Hz, H6a), 4.46 (dd, 1H, J5,6b = 4.4 Hz, Jgem = 11.9 Hz,
H6b), 4.36 (d, 1H, J1,2 = 10.1 Hz, H-1b), 3.56 (s, 3H, OCH3), 3.55 (m, 1H, H-5),
3.51 (dd, 1H, not assigned), 3.32–3.25 (2dd, 2H, not assigned).(d) Compound
(data not shown). The best
a-selectivities were obtained with
MeOTf in diethyl ether at ambient temperatures (0–25 °C) forming
17a13b in moderate yields. Despite the non-stereoselective reac-
tion, the two stereoisomers could be separated with ease via silica
gel column chromatography. Further improvements to enhance
the stereoselectivity might be possible by examining different
leaving groups (e.g., sulfoxides,12d trichloroacetimidates12e or
phosphates12f) or the use of more powerful promoters (e.g., N-
16d (CDCl3); 1H NMR: d = 5.81, 5.67 (2d, 2H, J = 2.6 Hz, H-1 , H-1a0), 5.47 (dd,
a
2H, J3,4ꢀJ4,5 = 9.8 Hz, H-4, H-40), 4.96, 4.70 (2d, 4H, Jgem = 11.1 Hz, CH2-2NM),
4.19 (m, 2H, H-5, H-50), 4.17 (dd, 2H, J2,3 = 9.8 Hz, H-3, H-30), 3.68, 3.62 (2dd,
2H, J2,3 = 10.1 Hz, J1,2 = 2.6 Hz, H-2, H-20), 3.54 (dd, 2H, J = 2.6 Hz, Jgem = 11.0 Hz,
H-6a, H-6a0), 3.44 (dd, 2H, J = 6.0 Hz, H-6b, H-6b0), 3.31 (s, 6H, 2 ꢂ OCH3).
11. (a) Pizey, J. S. Synthetic Reagents; Wiley: New York, 1974; Vol. 2, pp 65–142.; (b)
Hudlicky, M. J. Org. Chem. 1980, 45, 5377; (c) Shioiri, T.; Aoyama, T.; Mori, S.
Org. Synth. 1990, 68, 1; (d) Ohno, K.; Nishiyama, N.; Nagase, H. Tetrahedron Lett.
1979, 20, 4405; (e) Aoyama, T.; Shioiri, T. Tetrahedron Lett. 1990, 31, 5507; (f)
Chavis, C.; Dumont, F.; Wightman, R. H.; Ziegler, J. C.; Imbach, J. L. J. Org. Chem.
1982, 47, 202.
(phenylthio)-
The reduction of the azido group in 17
e
-caprolactam12g).
a
was attempted with Zn
under mild acidic conditions (step i) to produce 2-amino com-
pound 18. The choice of a proper protected GABA-synthon was cru-
cial for coupling to 18. Initially, we attempted to use commercially
available, N-protected GABA synthons Fmoc-GABA,8b Boc-GABA8c
and DTPM-GABA,14 but only low coupling yields were obtained
12. (a) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990,
31, 1331; (b) Loenn, H. Carbohydr. Res. 1985, 139, 105; (c) Loenn, H. Carbohydr.
Res. 1985, 139, 115; (d) Yan, L.; Kahne, D. J. Am. Chem. Soc. 1996, 118, 9239. and
references cited therein; (e) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212; (f) Plante, O. J.; Andrade, R. B.; Seeberger, P. H. Org. Lett. 1999, 1, 211; (g)
Duron, S. G.; Polat, T.; Wong, C.-H. Org. Lett. 2004, 6, 839.
(data not shown). Therefore, c-azido butyric acid 6 was selected
as the side chain synthon, which offers the advantage of releasing
the amino group under mild reaction conditions. As expected, the
DCC mediated coupling (step j) proceeded in a rapid and clean
fashion to afford the product 19 in high yield after normal phase
purification. The deprotection of 19 to the target molecule was
achieved in two steps. In the first step, the two benzoyl moieties
were removed via LiOH mediated saponification (step k) under
aqueous conditions to furnish compound 20 in high yield. The final
13. Selected NMR data of intermediates; 1H NMR were recorded at 400 MHz, 13C
NMR at 100 MHz; the solvent is mentioned in brackets after the compound
number,(a) Compound 17b (CDCl3); 1H NMR: d = 5.25 (dd, 1H, J3,4ꢀJ4,5 = 9.3 Hz,
H-4), 4.92 (d, 1H, Jgem = 11.6 Hz, CH2-2NM), 4.76 (d, 1H, Jgem = 11.6 Hz, CH2-
2NM), 4.38 (d, 1H, J1,2 = 7.3 Hz, H-1 b), 3.36 (s, 3H, OCH3).(b) Compound 17
a
(CDCl3); 1H NMR: d = 5.30 (dd, 1H, J3,4ꢀJ4,5 = 9.6 Hz, H-4), 5.01 (d, 1H,
J1,2 = 3.4 Hz, H-1a), 4.93 (d, 1H, Jgem = 11.3 Hz, CH2-2NM), 4.76 (d, 1H,
target, ACL 21269, was obtained via reduction of the c-azido group
Jgem = 11.3 Hz, CH2-2NM), 4.13 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.6 Hz, H-3), 3.97
(m, 1H, -CH2), 3.84 (m, 1H, -CH2), 3.58 (m, 1H, H-5), 3.48 (dd, 1H,
under mild acidic conditions. The product was obtained after filtra-
tion, extractive workup and freeze drying as a white amorphous
solid. Its identity was confirmed via LCMS-analysis and NMR-
spectroscopy.15
a
a
J1,2 = 3.4 Hz, J2,3 = 9.8 Hz, H-2), 3.37 (dd, 1H, J5,6a = 2.7 Hz, J6a,6b = 10.8 Hz, H-6a),
3.32 (dd, 1H, J5,6b = 5.8 Hz, J6a,6b = 10.8 Hz, H-6b), 3.26 (s, 3H, OCH3), 3.04 (m,
2H, b-CH2). 13C NMR: d = 165.50, 165.44 (C@O), 97.82 (C-1), 36.10 (
30.04 (b-CH2).
a-CH2),
14. DTPM-
c-aminobutyric acid (DTPM-GABA) was prepared from 1 via oxidation
References and notes
as described in Scheme 2, step e (71%).
15. ACL 21269: m/z = 539.115 [M+1]+; 1077.228 [2M+1]+; 1H NMR (400 MHz,
CD3CN): d = 7.88–7.78 (m, 4H, NM), 7.52–7.43 (m, 3H, NM), 7.06 (t, 1H, H-5 Ph,
J1ꢀJ2 = 8.0 Hz), 6.73–6.70 (m, 2H, H-6 Ph, H-2 Ph), 6.62 (m, 1H, H-4 Ph), 5.97 (d,
1H, NH, J = 9.3 Hz), 4.92 (d, 1H, Jgem = 11.7 Hz, CH2-2NM), 4.76 (d, 1H,
1. Ferrara, N.; Kerbel, R. S. Nature 2005, 438, 967. and references cited therein.
2. (a) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Salvino, J.; Leahy, E. M.;
Arison, B.; Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.; Sprengeler, P. S.;
Hamley, P.; Smith, A. B., III; Reisine, T.; Raynor, K.; Donaldson, C.; Vale, W.;
Maechler, L.; Freidinger, R. M.; Cascieri, M. A.; Strader, C. D. J. Am. Chem. Soc.
1993, 115, 12550; (b) Wunberg, T.; Kallus, C.; Opatz, T.; Henke, S.; Schmidt, W.;
Kunz, H. Angew. Chem., Int. Ed. 1998, 37, 2503; (c) Sofia, M. J.; Hunter, R.; Chan,
T. Y.; Vaughan, A.; Dulina, R.; Wang, H.; Gange, D. J. Org. Chem. 1998, 63, 2802;
(d) Kallus, C.; Opatz, T.; Wunberg, T.; Schmidt, W.; Henke, S.; Kunz, H.
Tetrahedron Lett. 1999, 40, 7783.
Jgem = 11.7 Hz, CH2-2NM), 4.59 (d, 1H, H-1
J2,3 = 10.2 Hz, J1,2 = 3.5 Hz, JNH,2 = 9.9 Hz), 3.88 (m, 1H,
a
, J1,2 = 3.5 Hz), 3.95 (ddd, 1H, H-2,
-CHa), 3.61–3.45 (m,
a-CHb), 3.31 (s, 3H, OCH3), 2.79 (m, 2H, b-CH2),
a
6H, H-5, H-4, H-6a, H-6b, H-3,
2.59 (t, 2H, Jvic = 6.5 Hz, a0-CH2), 2.02 (m, 2H, c0-CH2), 1.53 (quin, 2H, J = 7.1 Hz,
b0-CH2). 13C NMR (100 MHz, CD3CN): d = 172.45 (C@O), 157.14 (C-3 Ph), 141.41
(C-1 Ph), 129.70 (C-5 Ph), 120.61 (C-6 Ph), 117.33 (C-2 Ph), 114.73 (C-4 Ph),
98.19 (C-1
5), 68.34 (
25.73 (c0-CH2), 25.06 (b0-CH2).
a
a
), 80.65 (C-3), 73.86 (CH2-NM), 72.72 (C-6), 71.49 (C-4), 70.53 (C-
-CH2), 59.65 (OCH3), 51.94 (C-2), 35.62 (b-CH2), 34.06 (a0-CH2),
3. (a) Becker, B.; Condie, G. C.; Thanh Le, G.; Meutermanns, W. Mini-Rev. Med.
Chem. 2006, 6, 1; (b) Thanh Le, G.; Abbenante, G.; Becker, B.; Grathwohl, M.;