Improved synthesis of 15-deoxyspergualin analogs using the Ugi multi-component reaction

Article abstract of DOI:10.1016/j.bmcl.2011.02.079

Spergualin is a natural product that exhibits immunosuppressive, anti-tumor and anti-bacterial activities. Its derivatives, such as 15-deoxyspergualin (15-DSG), have been clinically approved for acute allograft rejection. However, the reported syntheses a

Full text of DOI:10.1016/j.bmcl.2011.02.079

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2587–2590
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Bioorganic & Medicinal Chemistry Letters
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Improved synthesis of 15-deoxyspergualin analogs using the Ugi
multi-component reaction
⇑
Christopher G. Evans , Matthew C. Smith , James P. Carolan, Jason E. Gestwicki
Department of Pathology and The Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Spergualin is a natural product that exhibits immunosuppressive, anti-tumor and anti-bacterial activities.
Its derivatives, such as 15-deoxyspergualin (15-DSG), have been clinically approved for acute allograft
rejection. However, the reported syntheses are cumbersome (>10 steps) and they suffer from low overall
yields (ꢀ0.3% to 18%). Moreover, spergualin and its derivatives are chemically unstable and rapidly
hydrolyzed in aqueous buffer. Here, we have re-explored these issues and report a modified synthetic
route with significantly improved overall yield (ꢀ31% to 47%). The key transformation is a microwave-
accelerated Ugi multi-component reaction that is used to generate the peptoid core in a single step. Using
the products of this route, we found that modifications of the hemiaminal significantly increased
chemical stability. Thus, we anticipate that this synthetic route will improve access to biologically active
15-DSG derivatives.
Received 21 December 2010
Revised 18 February 2011
Accepted 22 February 2011
Available online 1 March 2011
Keywords:
Polyamine
Chemical stability
Combinatorial synthesis
Immunosuppression
Peptoid
Ó 2011 Elsevier Ltd. All rights reserved.
Antibacterials
Spergualin is a natural product derived from Bacillus laterospo-
rus, which has gathered significant medical interest because of its
potent immunosuppressive, anti-tumor and anti-bacterial
activities.1–4 Early structural studies revealed that spergualin is
composed of three key regions: a peptoid core, guanidylated alkyl
group and spermidine-derived polyamine ( Fig. 1)5 and each of
these modules are thought to be required for biologic activity.6–8
However, attempts to further characterize the pharmacology of
spergualin were complicated by its rapid hydrolysis in aqueous
buffers. Thus, a major goal of early synthetic efforts was to modify
the most labile regions, such as the hydroxyl at position 15.8 These
efforts yielded the significantly more stable (À)-15-deoxyspergua-
lin (15-DSG) (Fig. 1), which was granted clinical approval to treat
acute allograft rejection.9 More recent efforts have yielded addi-
tional derivatives, such as tresperimus (Fig. 1), in which a portion
of the unstable peptoid is replaced with a carbamate.10,11 However,
although these derivatives are an improvement on spergualin, they
are still relatively unstable, with short half-lifes (t1/2) in neutral
and basic conditions.10 In addition, they are only weakly orally bio-
available (<5%) and metabolized rapidly, with terminal half-lifes of
only 1–2 h.12
Figure 1. Chemical structures of the natural product, spergualin, and two of its
derivatives. A schematic of the architecture is shown and the positions of carbons
11 and 15 are indicated.
15-DSG in only 0.3% yield in more than 10 steps, starting from
One major challenge in the search for improved spergualin
derivatives is that the reported synthetic routes are cumbersome
and low yielding. For example, the first attempts produced
L
-lysine and 1-amino-propanol (Fig. 2A).6,7,13 Later efforts mildly
improved the yield of 15-DSG (from ꢀ7% to 18%) by starting with
7-bromoheptanenitrile and protected spermidine derivative
a
( Fig. 2B), but these convergent routes remain protracted (>10
steps) and challenging.14–16
We envisioned an alternative synthetic approach. Specifically,
our retrosynthetic analysis employs the Ugi multi-component
⇑
Corresponding author. Tel.: +1 734 615 9537; fax: +1 734 764 1247.
E-mail address: gestwick@umich.edu (J.E. Gestwicki).
These authors contributed equally.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.079

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C. G. Evans et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2587–2590
Figure 2. Comparison of the retrosynthetic analyzes. Known convergent routes to spergualin (A) and 15-DSG (B) proceed through a series of more than 10 convergent steps.
(C) The proposed route employs the Ugi multi-component reaction to generate the peptoid and guanidylated regions in a single step. See the text for references.
Table 1
reaction to assemble the core of spergualin in a single step (Fig. 2C).
The Ugi reaction proceeds through the condensation of a carboxylic
Optimization of the Ugi reaction conditions
acid, amine, isocyanide, and aldehyde (or ketone).17,18 Thus, in
addition to the potential advantages gained from the concise crea-
tion of the peptoid, this route is also expected to allow access to
combinatorial diversity through varying the identity of the four
modules, including the guanidinylated amino acid (1), protected
isocyanide (2) and aldehyde (3).
Many conditions for the Ugi reaction have been reported, with
Entry
Solvent
Temp (°C)
Time (min)
Yield (%)
the most recent studies focusing on the use of microwave irradia-
tion to improve yield and reaction times.19,20 Guided by these ef-
forts, we first tested the feasibility of the approach using the
commercially available isocyanide, 1-isocyanopentane, as a model
reactant. Briefly, an equimolar solution of benyzlamine, benzalde-
hyde, 6-guanidinoheptanoic acid,21 and 1-isocyanopentane was
heated in methanol at 120 °C for 20 min in a microwave reactor.
These conditions yielded the product in good (70%) yield. Encour-
aged by this observation, we next substituted the simple isocya-
nide with the desired, tert-butyl (4-isocyanobutyl)carbamate
(2).22 Unfortunately, we found that this change dropped the yield
to 11%. To solve this issue, we re-investigated the reaction condi-
tions, focusing on the variables of time, temperature, and solvent
(Table 1). This effort revealed that the yield was particularly sensi-
tive to solvent and that DMF tended to be the best choice (average
purified yields between 36% and 46%).
1
2
3
4
5
6
7
8
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
EtOH
DMF
DMF
DMF
100
100
90
20
30
20
30
20
10
10
240
20
40
20
20
240
2–10
<2
36
22
<2
12
nd
35
27
17
46
36
43
90
110
120
130
rt
90
90
100
120
rt
9
10
11
12
13
Using the optimized Ugi conditions (DMF, 100 °C, 20 min), we
then pursued the synthesis of 15-DSG derivatives. Although this

C. G. Evans et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2587–2590
2589
reaction is often used in the assembly of large, combinatorial
libraries, our first efforts focused on producing a small number of
focused derivatives to test specific issues related to chemical sta-
bility (see below). Towards that goal, we combined four aldehydes
(3a–d) with 7-guanidinoheptanoic acid (1), benzylamine, and
tert-butyl (4-isocyanobutyl)carbamate (2) to produce intermedi-
ates 4a–d after Boc deprotection. In the next step, we envisioned
using a reductive amination to complete the sperimidine module.
Towards that goal, we first assembled Fmoc-3-amino-1-propanal
(5) from Fmoc-b-alanine, using the method of More and Finney.23
Then, compound 5 (0.05 mmol, 1 equiv) was reacted with 4a–d
(0.05 mmol, 1 equiv) in 2 mL DCE and NaBH(OAc)3 (0.07 mmol,
1.4 equiv) for 1.5 h to produce intermediates 6a–d. Importantly,
NaBH(OAc)3 was chosen for this step because, unlike NaBH3CN, it
does not require low pH.24 This was an important consideration
because of the sensitivity of spergualin analogs to degradation.
Following the reductive amination, we sought to remove the final
protecting groups. However, we found that common ways of
removing Fmoc, such as 20% piperidine in DMF, caused significant
hydrolysis. Therefore, we employed the alternative, Tris-amine
resin (50 equiv) in CHCl3 for 20 min.25 Finally, the benzyl group
was readily removed under mild conditions using ammonium cer-
ium (IV) nitrate (CAN).26 Following a final purification by alumina
chromatography, the products 7a–d were isolated as racemic
mixtures in 31–47% overall yield (Fig. 3).
With these compounds in hand, we wanted to examine their
stability in aqueous buffers. As mentioned above, one of the biggest
challenges in studies of spergualin and its analogs is that they are
prone to hydrolysis. Previous work had suggested that removal of
the labile 15-hydroxyl group could improve stability.11 However,
Lebreton et al. found that even the clinically approved 15-DSG
had a t1/2 of only ꢀ2 h in pH 10 buffer.10 Under these conditions,
degradation is thought to occur via hydrolysis at the hemiaminal
(Fig. 4C).27 Thus, we reasoned that blocking this degradation route
by modifying the C11 position might improve persistence. To test
this idea, we first compared the stability of spergualin and
11-methoxy-15-deoxyspergualin (7a) using thin layer chromatog-
raphy. Consistent with previous findings, the natural product
(Sigma Aldritch cat #S5822) is relatively stable under mild acidic
conditions (pH 5.0) but it is highly unstable in neutral and basic
buffers (Fig. 4A). By comparison, compound 7b, with the methoxy
substitution at position C11, had significantly improved stability
under basic and neutral conditions (Fig. 4B). Next, we quantified
the t1/2 values of compounds 7a–d and compared them to those
of spergualin and 15-DSG. In these studies, we found that the t1/2
values of compound 7a at pH 7.0 and 8.0 were 8- and 120-fold
greater than spergualin and at least 4-fold greater than 15-DSG
(Fig. 4D). Even the relatively simple substitution in 7d, removal
of the C11 hydroxyl, greatly improved stability. More striking,
bulky substitutions at position 11, as in compound 7b and c,
greatly improved stability to greater than 2 weeks. Together, these
results suggest that multiple types of substitutions at the hemia-
minal could provide significant increases in stability under neutral
and basic conditions.
To understand whether the introduced substitutions might
disrupt bioactivity, we tested compounds 7a–d in a series of
anti-bacterial assays against Escherichia coli, Bacillus subtilis and
Staphylococcus aureus. Consistent with previous reports, spergualin
and 15-DSG had anti-bacterial activity, especially against the
gram-positive strains (Fig. 4D). We found that compounds 7a–d
retained some anti-bacterial activity, although the relative poten-
cies were decreased. Of these compounds, 7d appeared the most
promising, with good activity against B. subtilis and S. aureus.
Additional studies are clearly needed to understand the relevant
structure–activity relationships and to improve compound po-
tency. However, these initial efforts show that spergualin analogs
with greatly improved chemical stability retain some bioactivity.
In this work, an improved synthetic method for producing
15-DSG analogs was developed, which increased the overall yields
by at least 2-fold and greatly reduced the number of steps. The key
transformation is the Ugi reaction to simultaneously generate the
peptoid and guanidylated regions. Using this approach, we found
that substitutions of the hemiaminal significantly improved chem-
ical stability which is expected to provide a path towards explora-
tion of these compounds as both research probes and therapeutics.
Figure 3. Synthesis of spergualin analogs. The Ugi condensation of aldehydes 3a–d, followed by Boc deprotection, produced the intermediates 4a–d in good yield. Reductive
amination with the aldehyde 5, followed by deprotection of the final Fmoc and benzyl groups yielded the final products 7a–d in overall, purified yields from 31% to 47%.

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C. G. Evans et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2587–2590
Figure 4. Synthetic derivatives of spergualin are more stable than the natural product. (A) The stability of spergualin was tested by thin layer chromatography. The time
points were initiated immediately after dissolution in aqueous buffers at the indicated pH. (B) Stability of 7a under the same conditions. Results are the average of at least
independent triplicates and the error bars represent standard error of the mean. (C) Summary of the known hydrolysis products of 15-DSG (D) Table of the stability values for
spergualin, 15-DSG and compounds 7a–d. Also included are the relative anti-bacterial activities (+++MIC <10
ÀMIC >250 g/mL).
lg/mL; ++ MIC 10–50 lg/mL; +MIC 50–250 lg/mL;
l
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Acknowledgments
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C.G.E. was supported by a predoctoral fellowship from the Cel-
lular Biotechnology Training Grant (GM008353). M.C.S. was sup-
ported by
a predoctoral fellowship from the Biogerontology
Training Grant (AG000114). This work was additionally supported
by grants from the NIH (NS059690) and NSF (MCB-0844512).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.079.
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