Preparation and separation of antimicrobial agents derived from capreomycin

Article abstract of DOI:10.1016/j.tetlet.2010.04.136

A practical method for the preparation of single component biaryl-urea analogs of capreomycin is described. The protocol involves derivatization of capreomycin followed by global protection of the capreomycin analog mixture. The individual components were separated using simple flash column chromatography followed by deprotection to give the desired analogs with high purity.

Full text of DOI:10.1016/j.tetlet.2010.04.136

Tetrahedron Letters 51 (2010) 3709–3711
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Tetrahedron Letters
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Preparation and separation of antimicrobial agents derived from capreomycin
1
*
D. David Hennings , Daniel J. Watson, Joe P. Lyssikatos , Andrew Allen
Array BioPharma Inc., Boulder, CO 80503, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A practical method for the preparation of single component biaryl-urea analogs of capreomycin is
described. The protocol involves derivatization of capreomycin followed by global protection of the cap-
reomycin analog mixture. The individual components were separated using simple flash column chroma-
tography followed by deprotection to give the desired analogs with high purity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 29 March 2010
Revised 26 April 2010
Accepted 28 April 2010
Available online 20 May 2010
Various analogs of the capreomycins have been prepared
semi-synthetically and have shown unique antimicrobial activity.1
Capreomycin, isolated by Herr and co-workers at Lilly2 from fer-
mentations of Streptomyces capreolus, is a mixture of four compo-
nents; capreomycins IA (1a) and IB (1b) comprise ꢀ90% of the
mixture and capreomycins IIA (1c) and IIB (1d) account for ꢀ10%
of the mixture (Fig. 1). We were interested in preparing biaryl-urea
analogs of capreomycin which exhibited potent in vitro activity
against MRSA (Methicillin-Resistant Staphylococcus aureus) and
other Gram positive bacteria. Utilizing the previously reported ure-
ido exchange reaction,3 our colleagues had determined that a mix-
ture comprised primarily of the two analogs shown in Figure 1,
AR380559 (2a) and AR380547 (2b), displayed potent in vitro activ-
ity against MRSA as well as other Gram positive bacteria.4
of constituents present in the starting capreomycin sulfate
(1a–d), we required a scalable semi-synthetic process that would
provide the single components in high purity. To achieve this goal
we either needed to purify the initial capreomycin sulfate mixture
to give one individual starting material or develop a method to
purify the product mixture. The isolated product mixture from
the ureido exchange reaction (primarily comprised of 2a and 2b)
exhibits similar physical properties to capreomycin sulfate,
namely, it is an amorphous powder with low organic solubility.
After encountering significant issues developing an analytical HPLC
method to separate 2a and 2b it was determined that preparative
HPLC was not viable. Furthermore, simple flash chromatography of
either the initial capreomycin sulfate mixture (1a–d) or the biaryl-
urea mixture was not pragmatic due to the similar retention times
observed for the individual components. In turn, we looked to mod-
ify the product mixture using protecting groups to give favorable
Although the ureido exchange reaction proceeds quite cleanly
giving a similar ratio of capreomycin analogs relative to the ratio
Figure 1. Capreomycin constituents and related analogs.
* Corresponding author. Tel.: +1 303 386 1221; fax: +1 303 381 6662.
E-mail address: dhennings@arraybiopharma.com (D.David Hennings).
Present address: Genentech, 1 DNA Way, MS 232A, South San Francisco, CA 94080-4990, United States.
1
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.136

3710
D. David Hennings et al. / Tetrahedron Letters 51 (2010) 3709–3711
R
O
O
O
O
NHBoc
H
N
BocHN
NHBoc
N
N
H
1. 2M HCl, MeCN
65 ºC, 4h
H
O
O
NH
HN
Capreomycin sulfate
+
H
N
H
H
N
H2N
N
H
2. Boc2O (3 eq)
Na2CO3 (4 eq)
MeCN, RT, 20h
(1a-d)
O
H
HN
O
HN
3-5 eq.
(~90% yield of 3a & 3b)
HN
N
H
3a; R = OH
3b; R = H
Scheme 1. Ureido exchange reaction and Boc protection.
physical properties that would allow us to separate the mixture of
2a and 2b. The most obvious solution was to exploit the serine
handle of 2a as a means of separation, via either crystallization, so-
lid-phase capture of the hydroxyl moiety or chromatography.
Conversion of capreomycin to the biaryl-urea analogs was per-
formed employing the ureido exchange reaction using similar con-
ditions to that previously reported1a for the preparation of phenyl
urea analogs (Scheme 1). After screening various co-solvents (diox-
ane, MeOH, EtOH, IPA, MeCN, and THF) and acids (HCl, H2SO4,
H3PO4, and HOAc) we determined that an equal mixture of 2 N
HCl and MeCN was the optimal solvent system for conversion of
capreomycin to the desired biaryl ureas. As this is a reversible pro-
cess, it was observed that using an excess of 1-(biphenyl-3-yl)urea
favored formation of the desired products. Under optimal reaction
conditions we determined that it was necessary to employ 3–
5 equiv of the urea to obtain >90% conversion to the ureido ex-
change products. The products could be isolated using the resin
capture strategy previously reported,1a however, we were able to
employ an extractive protocol for isolation of the product mixture.5
Upon reaction completion, the unreacted biaryl urea was removed
(and recycled) by extraction using EtOAc and the product-rich
aqueous layer was lyophilized to give the product mixture as a
white powder. Treatment of the crude mixture with di-tert-bu-
tyl-dicarbonate (Boc2O) gave quantitative conversion of all capreo-
mycin-related components to the globally protected analogs which
could be extracted into EtOAc and concentrated to give a mixture
composed primarily of compounds 3a and 3b.
Attempts to separate 3a from 3b by simple crystallization or so-
lid-phase capture using a resin technology proved fruitless. We
discovered, after evaluating hydroxyl-protecting groups including
acylation with acid chlorides and formation of silyl ethers, that
protection of the serine hydroxyl moiety with a silyl group suffi-
ciently altered the polarity of the IA and IB components so that
they could be separated using simple flash column chromatogra-
phy. An additional benefit of a silyl ether-protecting group was
that complete deprotection of the IA component could potentially
be accomplished in the same step as deprotection of the Boc-pro-
tected amines. In order to determine the most practical silyl ether
for this purpose, a screen was performed utilizing several commer-
cially available silyl chlorides (Table 1). To evaluate the polarity
difference between various silyl ethers the 3a/b mixture was re-
acted with the silyl chloride identified in Table 1 and the crude
reaction mixtures were analyzed using a trivial HPLC method.6
The difference in retention times between 4a–e and 3b was then
used as an indication of the polarity difference between the silyl
ether analog and 3b. Based on the results summarized in Table 1,
the TBDPS group (entry 4a) gave the greatest difference in reten-
tion time indicating that it would be most easily separated from
3b. Additionally, the TBDPS ether was sufficiently stable to enable
a chromatographic separation to be executed and therefore it was
chosen for preparative use (Scheme 2). The overall yield (of both
congeners) for the analog formation, protection, and chromato-
graphic separation of the two main components (4a and 3b) was
46%.5
The desired single component products were easily isolated
after removal of the protecting groups (Scheme 3). Deprotection
of 3b was accomplished using anhydrous HCl in MeOH to cleanly
give the tetra-HCl salt of AR380547 (2b) in 94% yield.7 Similarly,
treatment of 4a with HF in methanol followed by anhydrous HCl
in methanol cleanly liberated a mixed HF/HCl salt of AR380559
(2a) in 90% yield.8
In conclusion, this protocol provides a synthetically useful and
potentially scalable method for the preparation of single compo-
nent biaryl-urea capreomycin analogs such as AR380559 (2a) and
Table 1
Screen of silyl-protecting groups
Entry
HPLC retention times for various analogs (min)
Silyl chloride
IA silyl ether
IIB analog (3b)
Difference
4a
4b
4c
4d
4e
tBuPh2SiCl
Ph2MeSiCl
Ph3SiCl
tBuMe2SiCl
Et3SiCl
3.46
3.24
3.44
3.16
3.15
2.57
2.63
2.63
2.65
2.62
0.89
0.61
0.81
0.51
0.53
OTBDPS
H
O
O
O
NHBoc
O
O
O
NHBoc
H
N
1. TBDPSCl
BocHN
N
N
NHBoc
BocHN
NHBoc
imidazole
N
N
H
N
H
DMF, RT, 19h
H
H
O
O
O
O
H
3a/3bmixture
NH
HN
NH
HN
H
N
H
H
N
H
N
H
2. chromatography
+
N
O
H
HN
O
H
O
HN
HN
O
HN
HN
HN
N
H
N
H
4a
(22% from capreomycin)
3b
(24% from capreomycin)
Scheme 2. Protection with TBDPS and chromatographic separation.

D. David Hennings et al. / Tetrahedron Letters 51 (2010) 3709–3711
3711
Scheme 3. Deprotection of biaryl analogs.
reduced pressure to give 11.5 g of a light yellow oil which was used directly in
the next step without purification. The oil was dissolved in 60 mL of DMF and
treated with imidazole (2.0 g, 29 mmol) and TBDPSCl (5.6 g, 20 mmol). The
mixture was stirred under N2 at ambient temperature for 19 h. The reaction
mixture was partitioned between 5% NaCl solution and EtOAc (400 mL each) and
the aqueous layer was extracted with EtOAc (200 mL). The combined organic
phases were washed with 0.1 N HCl, 10% NaHCO3, brine, and saturated NaCl
solution (150 mL each), dried over MgSO4, and concentrated under reduced
pressure to give a foam. The foam was purified by flash chromatography on
silica gel (elution with 0–15% MeOH in dichloromethane) to give 3.7 g (24%) of
3b (Rf = 0.23 in 10% MeOH in DCM) and 4.1 g (22%) of 4a (Rf = 0.44 in 10% MeOH
in DCM), both as white powders.
AR380547 (2b) in good yields and high purities using standard lab-
oratory techniques.
Acknowledgments
We thank Dr. Lingyang Zhu for assistance with NMR analysis,
Dawn Cherico for HPLC method development, and Cale Bibb and
Dr. Phil Anderson for providing HRMS analysis.
6. HPLC conditions: YMC ODS-AQ 4.6 Â 50 mm S-5
l 120 Å column; eluent
Supplementary data
A = water with 1% IPA and 0.01% HFBA (heptafluorobutyric acid); eluent
B = MeCN with 1% IPA and 0.01% HFBA; 4 min gradient with 5% to 95% B; flow
rate = 2 mL/min.; UV detection at 254 nm.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.04.136.
7. Procedure for preparation of 2a: To a solution of 4a (0.53 g, 0.39 mmol) in MeOH
(5 mL) was added concentrated aqueous HF (0.57 mL, 15.6 mmol) and the
mixture was stirred at 60 °C for 30 min and then cooled to ambient temperature.
A solution of 3.0 M anhydrous HCl in MeOH (5.2 mL, 15.6 mmol) was added to
the reaction mixture and the resulting mixture was stirred at ambient
temperature overnight. The vessel was opened to the atmosphere and heated
until most of the MeOH had been removed. The resulting solid was triturated
with MTBE (20 mL) and the product was isolated by vacuum filtration and dried
to give 0.34 g (90%) of 2a as a white powder. 1H NMR (500 MHz, D2O) d 1.59 (dt,
J = 14.6, 14.3, 8.6, 6.8 Hz, 1H) 1.67 (br m, 4H), 1.97 (dt, J = 14.6, 5.5, 5.1 Hz, 1H),
2.55 (dd, J = 16.4, 8.3 Hz, 1H) 2.67 (dd, J = 16.4, 4.6 Hz, 1H), 2.90 (br m, 2H), 3.13
(dd, J = 13.7, 9.6 Hz, 1 H), 3.22 (br t, J = 6.8, 5.5 Hz, 2H), 3.56 (br dt, J = 13, 4.7 Hz,
1H), 3.66 (d, J = 7.2 Hz, 2H,), 3.74 (d, J = 5.2 Hz, 1H), 3.98 (dd, J = 13.7, 5.2 Hz, 1H),
4.21 (dd, J = 9.6, 5.2 Hz, 1H), 4.30 (t, J = 7.2 Hz, 1H), 4.34 (ddd, J = 8.6, 5.5, 2.4 Hz,
1H), 4.69 (t, J = 5.1 Hz, 1H), 4.91 (d, J = 2.4 Hz, 1H), 7.28 (br m, 1H), 7.34 (t,
J = 7.8 Hz, 1H), 7.39–7.37 (m, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.56 (m, 1H), 7.58 (d,
J = 8.0 Hz, 2H), 8.03 (br s, 1H); 13C NMR (125 MHz, D2O) d 22.8, 22.8, 28.9 36.1,
36.5, 38.0, 38.8, 39.4, 48.3, 53.4, 49.0, 50.9, 54.5, 54.9, 62.0, 105.1, 119.2, 119.9,
123.2, 126.9, 127.9, 129.1, 129.8, 134.7, 137.4, 139.9, 141.5, 153.0, 154.3, 167.0,
167.2, 171.0, 171.6, 172.0, 172.3; HRMS calcd m/z for C37H53N14O8 821.4165
(M+H)+, found 821.4185.
References and notes
1. (a) Dirlam, J. P.; Belton, A. M.; Birsner, N. C.; Brooks, R. R.; Chang, S.-P.;
Chandrasekaran, R. Y.; Clancy, J.; Cronin, B. J.; Dirlam, B. P.; Finegan, S. M.;
Froshauer, S. A.; Girard, A. E.; Hayashi, S. F.; Howe, R. J.; Kane, J. C.; Kamicker, B.
J.; Kaufman, S. A.; Kolosko, N. L.; LeMay, M. A.; Linde, R. G., II; Lyssikatos, J. P.;
MacLelland, C. P.; Magee, T. V.; Massa, M. A.; Miller, S. A.; Minich, M. L.; Perry, D.
A.; Petitpas, J. W.; Reese, C. P.; Seibel, S. B.; Su, W.-G.; Sweeney, D. A.; Whipple,
D. A.; Yang, B. V. Bioorg. Med. Chem. Lett. 1997, 7, 1139; (b) Linde, R. G., II; Birsner,
N. C.; Chandrasekaran, R. Y.; Clancy, J.; Howe, R. J.; Lyssikatos, J. P.; MacLelland,
C. P.; Magee, T. V.; Petitpas, J. W.; Rainville, J. P.; Su, W.-G.; Vu, C. B.; Whipple, D.
A. Bioorg. Med. Chem. Lett. 1997, 7, 1149.
2. Herr, E. B.; Haney, M. E.; Pittenger, G. E.; Higgins, C. E. Proc. Ind. Acad. Sci. 1960,
69, 134.
3. Nomoto, S.; Shiba, T. J. Antibiot. 1977, 30, 1008.
4. (a) Lyssikatos, J. P.; Wenglowsky, S. M.; Hennings, D. D.; Watson, D. J. WO 2007/
130743, 2007.; (b) Wenglowsky, S.; Lyssikatos, J.; Brown, S.; Hennings, D.;
Watson, D.; Winkler, J.; Lee, P.; Rieger, R.; Allen, S. Capreomycin Derivatives and
their Activity against Gram Positive Bacteria Including Methicillin Resistant S.
Aureus, 19th International Symposium on Medicinal Chemistry, Istanbul, Turkey,
August 29, 2006.
5. Procedure for preparation of 3b and 4a: To a mixture of capreomycin sulfate
(10.0 g, 13.5 mmol) and 3-biphenylurea (11.2 g, 52.7 mmol) was added 100 mL
of acetonitrile followed by 100 mL of freshly prepared 2 N HCl. The mixture was
then heated at 65 °C with stirring for 24 h. The reaction mixture was cooled to
room temperature and neutralized (pH 8) using 2.5 N NaOH solution. The
mixture was filtered to recover unreacted 3-biphenylurea and the filtrate was
washed with EtOAc (2 Â 100 mL). The product-rich aqueous layer was
lyophilized to give 22.2 g of an off-white powder. The solid was suspended in
200 mL of MeCN and treated with Boc2O (8.9 g, 41 mmol) and Na2CO3 (6.0 g,
56 mmol) and stirred at ambient temperature for 20 h. The reaction mixture was
diluted with 200 mL of water and extracted with EtOAc (2 Â 100 mL). The
combined organic phases were dried over MgSO4 and concentrated under
8. Procedure for preparation of 2b: To a solution of 3b (1.01 g, 0.91 mmol) in MeOH
(10 mL) was added a solution of 3.4 M HCl in MeOH (10 mL, 34 mmol) and the
mixture was stirred at ambient temperature for 3 h. The reaction mixture was
concentrated to give 0.82 g (94%) of 2b as a white powder. 1H NMR (500 MHz,
D2O) d 1.33 (d, J = 7.3 Hz, 3H), 1.62 (m, 1H), 1.70 (m, 2H), 1.70 (m, 2H), 1.97 (m,
1H), 2.56 (dd, J = 16.2, 8.7 Hz, 1H), 2.67 (dd, J = 16.8, 4.7 Hz, 1H), 2.94 (m, 2H),
3.24 (m, 2H), 3.25 (m, 1H), 3.55 (penta, J = 7.4 Hz, 1H), 3.61 (dd, J = 14.2, 10.5 Hz,
1H), 3.76 (dd, J = 14.7, 10.5 Hz, 1H), 4.04 (dd, J = 14.0, 5.1 Hz, 1H), 4.22 (dd,
J = 7.3, 4.6 Hz, 1H), 4.26 (m, 1H), 4.32 (q, J = 7.3 Hz, 1H), 4.32 (dd, J = 10.0, 5.6 Hz,
1H), 4.84 (dd, J = 2.4 Hz, 1H), 7.30 (m, 1H), 7.32 (m, 1H), 7.35 (m, 1H), 7.35 (m,
1H), 7.41 (m, 1H), 7.42 (m, 1H), 7.49 (d, J = 7.3 Hz, 1H), 8.01 (s, 1H); 13C NMR
(125 MHz, D2O) d 18.1, 22.9, 23.1, 29.3, 36.2, 36.6, 38, 39, 39.6, 48.3, 49, 49.2,
51.4, 53.4, 55.1, 105.4, 118.6, 119.5, 123.3, 127.0, 128.1, 129.2, 130, 134.8, 137.6,
139.9, 141.6, 152.1, 154.3, 166.8, 167.1, 171, 172, 172, 175.8. HRMS calcd m/z for
C
37H53N14O7 805.4216 (M+H)+, found 805.4233.