Design, synthesis and biological evaluation of simplified side chains of the macrolide antibiotic etnangien

Article abstract of DOI:10.1016/j.bmcl.2012.06.070

Novel simplified side chains of the potent RNA polymerase inhibitor etnangien were designed, synthesized and evaluated for antibacterial activity against Gram-positive bacteria and one Gram-negative bacterium.

Full text of DOI:10.1016/j.bmcl.2012.06.070

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5731–5734
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of simplified side chains
of the macrolide antibiotic etnangien
Mario Altendorfer a, Herbert Irschik b, Dirk Menche c,
⇑
a Organische Chemie, Ruprecht-Karls-Universität Heidelberg, INF 270, D-69120 Heidelberg, Germany
b Helmholtz-Zentrum für Infektionsforschung, Research Group Microbial Drugs, Inhoffenstr. 7, D-38124 Braunschweig, Germany
c Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel simplified side chains of the potent RNA polymerase inhibitor etnangien were designed, synthe-
sized and evaluated for antibacterial activity against Gram-positive bacteria and one Gram-negative
bacterium.
Received 24 May 2012
Revised 22 June 2012
Accepted 24 June 2012
Available online 14 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Etnangien
Analogues
RNA-polymerase
Structure–activity data
Antibiotics
The labile polyketide macrolide antibiotic etnangien (1, Fig. 1)
presents a highly potent antibiotic against bacteria, isolated from
various strains of the myxobacterium Sorangium cellulosum
So ce750 and So ce1045.1,2 It is a structurally unique type of a par-
ticularly efficient RNA polymerase inhibitor in vitro and in vivo3
with average IC50 values in the submicromolar scope.3 In detail,
it is effective against a broad panel of Gram-positive bacteria, espe-
cially those belonging to the actinomycetes.3 Notably, etnangien
(1) shows no cross-resistance to rifampicine, which presents the
only curative useful RNA-polymerase inhibitor so far. This qualifies
the bacterial RNA-polymerase as one of the rare validated, but
underexploited, targets for broad-spectrum antibiotics.4 Further-
more, etnangien (1) also retains activity against retroviral DNA
polymerase and shows low cytotoxicity against mammalian cells,
which add to its attractiveness for further investigation.3 The noto-
rious instability, of the side chain embodying a polyunsaturated
hexaene subunit demands the development of stable and more
readily available analogues to further advance this highly promis-
ing macrolide antibiotic.
etnangien (1) and its equipotent methyl ester derivative (2) have
been accomplished in our group.6,7 In connection with the total syn-
thesis a few simplified etnangien analogues have been obtained and
biologically evaluated against a range of Gram-positive and negative
bacteria.8
Herein, we report the synthesis of the first side chain analogues
of etnangien and their evaluation for antibacterial activity against
Gram-positive bacteria and one Gram-negative bacterium.
Inspired by various polyene antibiotics,9 we initiated a syn-
thetic programme developed towards polyene side chain ana-
logues of etnangien in a rationale to understand the biological
importance of this part of the etnangien structure. One focus was
placed on the design and synthesis of modified side chains, lacking
the notoriously labile polyene subunit. As shown in Scheme 1, our
concept relied on a late stage 3-fragment cross coupling strategy,
HO
OH
O
The full relative and absolute stereochemistry of etnangien (1)
has been determined in our group in a cooperation with the group
of Rolf Müller by a combination of extensive high-field NMR-studies,
modeling, chemical derivatization and an innovative bioinformatics
approach.5 Furthermore, a first and so far only total synthesis of
OH
O
OH
RO
O
OH
OMe
OH
1: Etnangien (R = H)
2: Etnangien methylester (R = Me)
⇑
Corresponding author. Tel.: +49 (0)228 732653; fax: +49 (0)228 735813.
E-mail address: dirk.menche@uni-bonn.de (D. Menche).
Figure 1. Etnangien (1) and its methyl ester (2).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.070

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M. Altendorfer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5731–5734
labile precursor molecule by installing the polyene fragment at a
Suzuki Coupling
very late stage. Also, replacement of the central bis-metallated
fragment enables a rapid access to various analogues.
O
OMe
As a first target, we decided to synthesize dienyl iodide 7. This
was available from the commercially available acid 8 in a six step
reaction sequence (Scheme 2), involving acid catalyzed esterifica-
tion, cross metathesis with crotonaldehyde using Hoveyda Grubbs
II catalyst, Brown allylation of the terminal allylic aldehyde,10 TBS
protection, another second cross metathesis croton aldehyde and
a subsequent Takai homologation.11 This sequence afforded vinyl
iodide 7 in high stereoselectivities and good yields (45% yield, 6
steps). Light sensitive dienyl iodide 7 was then immediately con-
nected to the bis-metallated central building block 5 by a Stille cou-
pling.12 Accordingly, treatment of iodide 7 and diene 5a with
PdCl2(CH3CN)2 in degassed DMF afforded the desired pinacolborane
8a in good yield. The analogous Stille coupling of stannane 5b and
dienyl iodide 7 under same conditions proceeded in moderate yield
generating methyl ester 8b. To complete the coupling sequence we
next focused on the construction of western fragments 6. The pri-
mary alcohol function of 9 was initially protected both as a silyl
and a PMB ether. Starting from aldehyde 9a we advanced towards
(E)-vinyl iodide 6a via asymmetric alkyne addition of TMS-acety-
lene13 to aldehyde 9a as a first key step. After TBS protection of
the selectively installed hydroxy group TMS was removed and the
terminal alkyne underwent a hydrozirconation reaction14 followed
by iodonolysis15 to obtain bis-TBS ether 6a (55% yield, 4 steps).
Using the same reaction conditions PMB ether 9b was readily trans-
formed into vinyl iodide 6b in 47% yield over 4 steps. Subsequently,
pinacolboranes 8 and iodides 6 underwent a Suzuki–Miyaura
coupling16 applying mild reactions conditions with Pd(dppf)Cl2 as
catalyst and Ba(OH)2Á8H2O as a base.17
n
RO
3a:
OH
OH
R = H, n = 0
3b: R = H, n = 1
Stille Coupling
4a: R = PMB, n = 0
4b:
R = PMB, n = 1
SnBu3
O
B
O
n
5a:
5b: n = 1
n = 0
O
I
I
OMe
RO
6a
OTBS
7
OTBS
: R = TBS
6b: R = PMB
Scheme 1. Late stage cross coupling strategy of simplified etnangien side chains.
O
O
a-f
OH
I
OMe
OMe
8
7
OTBS
g
SnBu3
O
B
O
n
5a
: n = 0
5b: n = 1
O
O
B
O
n
OTBS
I
8a
: n = 0
8b: n = 1
In total four different types of protected side chains could be
prepared demonstrating the general usefulness of our approach.
With the highly unsaturated polyenes 10a, 10b, 11a and 11b in
hand, efforts were then directed towards the challenging deprotec-
tion of these polyenes. After considerable experimentation, it was
found that best results in terms of yield and practicablity were ob-
tained with a protocol reported by Kishi,18 which involves treat-
ment with TBAF and anhydrous work-up procedure. In detail,
methyl esters 10a, 10b, 11a and 11b were treated with 1 M TBAF
and stirred at room temperature over night, resulted complete
and clean conversion. Finally, after working up with CaCO3, DOW-
EX and methanol the desired target compounds 3a, 3b, 4a and 4b
could be isolated in good yields.
Based on the syntheses of the simplified etnangien side chains
reported above, we decided to apply this 3-fragment coupling con-
cept also for the construction of the original side chain 12 of etnan-
gien. Again, we were focusing on avoiding unstable precursors
which allow an assembling of the labile hexaene subunit in an effi-
cient late stage cross coupling sequence (see Scheme 3).
The synthesis of the identical etnangien side chain 12 could
again be realized in a similar way to the above reported construc-
tion of simplified side chain derivatives 3 and 4, demonstrating
again the usefulness of this approach. Starting from aldehyde 9b
asymmetric addition13 of propyne afforded the desired propargylic
alcohol 16 in an anti Felkin–Anh preference (dr = 12:1). After TBS
protection of the installed secondary hydroxyl group, the triple
bond was transformed selectively into a stannylated olefin via
Palladium-catalyzed hydrostannylation.19 Finally, metal/halogen
exchange20 afforded vinyl iodide 14 in 27% over 4 steps. Subse-
quent Stille coupling12 of western fragment 14 with stannane 139
gave pentaene 17 in good yields considering the lablity of this
compound (67%). Next, the eastern fragment 15 was obtained in
reliable fashion by a 12 step procedure starting from commercially
available alcohol 18 (6.0% over 12 steps).6 As illustrated in Scheme
4, pinacolborane 17 and eastern fragment 15 underwent a Suzuki–
Me
Me
l
h-k
RO
OTBS
: R = TBS
RO
9a
O
6a
: R = TBS
6b: R = PMB
9b: R = PMB
O
OMe
OMe
n
RO
RO
OTBS
OTBS
10a: R = TBS, n = 0
10b
11a
: R = TBS, n = 1
: R = PMB, n = 0
11b: R = PMB, n = 1
m
O
n
OH
OH
3a
: R = H, n = 0
3b: R = H, n = 1
4a: R = PMB, n = 0
4b
: R = PMB, n = 1
Scheme 2. Synthesis of novel simplified etnangien side chains 3 and 4. Reagents
and conditions: (a) H2SO4, MeOH, reflux, 3 h, 72%; (b) crotonaldehyde, Hoveyda
Grubbs II, DCM, reflux, 2 h, 93%; (c) (À)-(Ipc)2B(allyl), Et2O, À100 °C, 90%, 96% ee; (d)
TBSOTf, 2,6-lutidine, DCM, À78 °C, 1 h, 99%; (e) crotonaldehyde, Grubbs II, DCM,
reflux, 2 h, 86%; (f) CHI3, CrCl2, THF/dioxane (1:6), 0 °C to rt, 87%, E/Z 7:1; (g)
PdCl2(CH3CN)2, DMF, rt, 12 h, 80% for 8a and 68% for 8b; (h) Et2Zn, TMS-acetylene,
toluene, reflux, 1 h then Ti(O-iPr)4, (R)-BINOL, Et2O, rt, 12 h, d/r = 8:1 using 9a and d/
r = 9:1 using 9b; (i) TBSOTf, 2,6-lutidine, DCM, À78 °C, 1 h; (j) K2CO3, MeOH, rt, 1 h;
(k) ZrCp2Cl2, DIBAL-H, THF, 30 min, À78 °C to rt then I2, À78 °C, 30 min, 55% over 4
steps for 6a and 47% for 6b; (l) Pd(dppf)Cl2, Ba(OH)2Á8H2O, DMF, rt, 4 h, 49% for 10a,
47% for 10b, 77% for 11a and 83% for 11b; (m) TBAF, THF, rt, 12 h then CaCO3,
DOWEX, MeOH, rt, 1 h, 59% for 3a, 95% for 3b, 27% for 4a and 61% for 4b.
involving a Stille/Suzuki–Miyaura coupling sequence bases on a
central bis-metallated reagent 5. Notably, this sequence avoids a

M. Altendorfer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5731–5734
5733
Table 1
Stille Coupling
OH
Antibiotic activity of etnangien analogues (3, 4, 12) in comparison to etnangien (1)
O
and its methyl ester (2)22
OMe
Test organsism
MICa
3a
(lg/mL)
PMBO
OH
12
13
1
2
3b
4a
4b
12
Suzuki Coupling
O
Staphylococcus aureus
Bacillus subtilis
C. glutanicum
1
10
2.5
20
>80
>80
>80
>80
>40
>80
>80
>80
80b
20
>80
>80
80b
>80
>40
>80
>80
>80
>80
>80
>80
>80
>40
>80
>80
>80
>40
>40
>40
>40
>40
>40
>40
>40
0.03
0.06
0.12
0.39
>80
>80
0.24
n. d.
n. d.
0.06
>20
>40
80b
40
C. mediolanum
B
Mycobacterium phlei
Micrococcus luteus
Escherichia coli
S. cerevisiae
>40
>80
>80
>80
O
Bu3Sn
I
O
I
OMe
a
Experiments were run in dublicates or triplicates.
Incomplete inhibition.
14
PMBO
OTBS
15
OTBS
b
Scheme 3. Late stage cross coupling strategy of the original etnangien side chain.
activity against Gram-negative E. coli and the yeast Saccharomyces
cerevisiae. However, a certain activity of triol 3b was observed
against Bacillus subtilis, with a similar potency as the parent natural
product 1 and its methyl ester derivative 2. Also a certain inhibi-
tion against Corynebacterium mediolanum was observed for 3b.
All other analogues, including the original side chain 12, showed
no or very low degrees of activities, suggesting a critical impor-
tance of the macrocyclic part for full biological potency.
In conclusion, we developed a highly concise first synthesis of
side chain analogues of the etnangiens enabling a first biological
evaluation of this structural part of these potent antibiotics. The
synthesis of these compounds was enabled by a highly modular
and convergent 3-fragment coupling strategy. Most side chain ana-
logues demonstrated little or no antibacterial potency, suggesting
that the macrocyclic core has to be present for full biological po-
tency. Together with previous data7 these results show that both
the macrocyclic core and the side chain are critical parts of the
pharmacophore. However, a certain degree of activity was also ob-
served for simplified side chain analogue 3b, suggesting that the
parent natural structure may still be simplified with retention of
activity.
Miyaura reaction16,17 to obtain bis-TBS ether 19 in high yield (83%).
Global deprotection using the established Kishi protocol18 afforded
the desired methyl ester 12 in 56% yield.21
The potent antimicrobial activity of etnangien motivated us to
likewise analyse the developed and more readily available side
chain analogues for their antibiotic potential. Table 1 presents their
inhibitory qualities against several microorganisms, in direct com-
parison to the authentic natural product etnangien (1) and its
methyl ester derivative (2). As previously reported, bacteria
belonging to the suborder of Corynebacterineae, such as Nocardia
coralline and some Mycobacteria, were particulary sensitive to (1)
and (2).
However, yeast and Gram-negative Escherichia coli proved to be
rather resistant. Presumably, this may be arised from the Gram-
negative character of myxobacteria.1,3 In agreement with these
data for the parent natural product 1 and its methyl ester 2, the
side chain analogues 3a, 3b, 4a, 4b and 12 likewise showed no
a
b-d
I
PMBO
OH
Acknowledgment
PMBO
O
PMBO
OTBS
9b
16
14
Generous financial support from the ‘Deutsche Forschungs-
gemeinschaft’ (Me 2756/4) is most gratefully acknowledged.
O
B
e
O
Bu3Sn
13
References and notes
O
B
1. Höfle, G.; Reichenbach, H.; Irschik, H.; Schummer, D. German Patent DE
1,953.098,0 A1: 1 (5.2.1998).
2. For a recent review on polyketides from myxobacteria, see Menche, D. Nat.
Prod. Rep. 2008, 25, 905.
O
17
PMBO
OTBS
3. Irschik, H.; Schummer, D.; Höfle, G.; Reichenbach, H.; Steinmetz, H.; Jansen, R. J.
Nat. Prod. 2007, 1060, 70.
4. (a) Darst, S. Trends Biochem. Sci. 2004, 29, 159; (b) Chopra, I. Curr. Opin. Invest.
Drugs 2007, 8, 600; (c) Haebich, D.; von Nussbaum, F. Angew. Chem. 2009, 121,
3447.
O
Ref. [6]
MeO
HO
f
I
5. Menche, D.; Arikan, F.; Perlova, O.; Horstmann, N.; Ahlbrecht, W.; Wenzel, S. C.;
Jansen, R.; Irschik, H.; Müller, R. J. Am. Chem. Soc. 2008, 130, 14234.
6. (a) Li, P.; Li, J.; Arikan, F.; Ahlbrecht, W.; Dieckmann, M.; Menche, D. J. Am.
Chem. Soc. 2009, 131, 11678; (b) Li, P.; Li, J.; Arikan, F.; Ahlbrecht, W.;
Dieckmann, M.; Menche, D. J. Org. Chem. 2010, 75, 2429.
7. For synthetic studies towards etnangien, see (a) Arikan, F.; Li, J.; Menche, D.
Org. Lett. 2008, 10, 3521; (b) Sabitha, G.; Yadagiri, K.; Bhikshapathi, M.;
Chandrashekhar, G. Tetrahedron. Asymmetry 2010, 21, 2524.
8. Menche, D.; Li, P.; Irschik, H. Bioorg. Med. Chem. Lett. 2010, 20, 939.
9. (a) Lee, S. J.; Anderson, T. M.; Burke, M. D. Angew. Chem. 2010, 122, 9044; (b)
Coleman, R. S.; Lu, X.; Modolo, I. J. Am. Chem. Soc. 2007, 129, 3826; (c) Sorg, A.;
Brückner, R. Angew. Chem. Int. Ed. 2004, 43, 4523.
10. (a) Racherla, U. S.; Brown, H. C. J. Org. Chem. 1991, 56, 401; (b) Racherla, U. S.;
Liao, Y.; Brown, H. C. J. Org. Chem. 1992, 57, 8614.
11. (a) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. So 1986, 108, 7408; (b) Okazoe,
T.; Takai, K.; Utimoto, K. J. Am. Chem. Soc. 1987, 109, 951.
12. Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508.
13. Marshall, J. A.; Bourbeu, M. P. Org. Lett. 2003, 5, 3197.
OTBS
15
19
O
18
MeO
OR
PMBO
OR
: R = TBS
12: R = H
g
Scheme 4. Synthesis of the original etnangien side chain 11. Reagents and
conditions: (a) Et2Zn, propyne, toluene, reflux, 1 h then Ti(O-iPr)4, (R)-BINOL,
Et2O, rt, 12 h, 60%, dr = 12:1; (b) TBSOTf, 2,6-lutidine, DCM, À78 °C, 1 h, 96%; (c)
Pd(Ph)3Cl2, n-Bu3SnH, THF, rt, 30 min, 68%; (d) I2, DCM, 0 °C, 15 min, 98%;(e)
PdCl2(CH3CN)2, DMF, rt, 12 h, 67%; (f) Pd(dppf)Cl2, Ba(OH)2Á8H2O, DMF, rt, 4 h, 83%;
(g) TBAF, THF, rt, 12 h then CaCO3, DOWEX, MeOH, rt, 1 h, 58%.

5734
M. Altendorfer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5731–5734
14. (a) Hart, D. W.; Blackburn, T. F.; Schwartz, J. J. Am. Chem. Soc. 1975, 97, 679; (b)
Wipf, P.; Jahn, H. Tetraheron 1996, 52, 12853.
15. Strand, D.; Rein, T. Org. Lett. 2005, 7, 199.
16. Suzuki, A. J. Organomet. Chem. 1999, 576, 147.
17. Fürstner, A.; Nevado, C.; Tremblay, M.; Chevrier, C.; Teply´, F.; Aïssa, C.; Waser,
(125.76 MHz, CDCl3): d = 13.6, 27.4, 32.4, 32.4, 33.6, 40.5, 41.0, 51.6, 67.6, 71.8,
78.6, 128.8, 129.7, 129.8, 130.6, 130.9, 131.9, 132.3, 132.3, 133.4, 133.9, 133.9,
134.9, 173.4; HR-MS (ESI): found m/z = 427.2459 (C24H3605Na), calculated m/
z = 427.2455. Compound 12: 1H NMR (600.130 MHz, CDCl3): d = 0.83 (d,
J=7.0 Hz, 3 H), 1.62 (br. s., 1H), 1.70 (s, 3H), 1.82 (s, 3H), 1.94 (m, 1H), 2.33
(m, 4H), 2.45 (m, 2H), 3.41 (br s, 1 H), 3.46 (t, J = 8.5 Hz, 1H), 3.59 (dd, J = 9.2 Hz,
J = 4.0 Hz, 1H), 3.67 (s, 3H), 3.81 (s, 3 H), 4.41 (m, 2 H), 4.47 (s, 2H), 5.22 (d,
J = 8.6 Hz, 1H), 5.48 (d, J = 8.9 Hz, 1H), 5.68 (m, 1H), 6.02 (m, 1H), 6.25 (m, 8H),
6.89 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H); 13C NMR (150.90 MHz, CDCl3):
d = 13.0, 13.4, 16.7, 32.5, 34.3, 39.2, 41.2, 51.6, 55.3, 68.0, 72.8, 73.1, 74.5, 113.8,
127.6, 128.7, 129.4, 129.8, 130.0, 132.0, 132.7, 132.9, 133.0, 133.1, 133.4, 133.8,
133.8, 136.1, 137.1, 137.4, 159.3, 173.6; HR-MS (ESI): found m/z = 573.3189
(C34H4606Na), calculated m/z = 573.3187.
M. Angew. Chem., Int. Ed. 2006, 45, 5837.
18. Kaburagi, Y.; Kishi, Y. Org. Lett. 2007, 9, 723.
19. Zhang, H. X.; Guibe, F.; Balavoine, G. J. Org. Chem. 1857, 1990, 55.
20. Takao, K.; Hayakawa, N.; Yamada, R.; Yamaguchi, T.; Morita, U.; Kawasaki, S.;
Tadano, K. Angew. Chem., Int. Ed. 2008, 47, 3426.
21. All final compounds reported in the paper were chromatographically purified
and characterized by NMR and HRMS. Characterization data for compound 3a:
1H NMR (500.132 MHz, CDCl3): d = 0.85 (d, J = 6.9 Hz, 2H), 1.65 (br s, 3H), 1.82
(ddt, J = 14.5 Hz, J = 10.8 Hz, J = 7.3 Hz, J = 7.3 Hz, 1 H), 2.34 (m, 10H), 3.65 (m,
1H), 3.68 (s, 3H), 3.75 (dd, J = 10.8 Hz, J = 3.4 Hz, 1 H), 4.05 (t, J = 7.7 Hz, 1H),
4.13 (dt, J = 11.9 Hz, J = 5.8 Hz, 1H), 5.63 (m, 6H), 6.12 (m, 6H); 13C NMR
22. MIC values were determined by serial dilution, for further information see:
Irschik, H.; Jansen, R.; Gerth, K.; Höfle, G.; Reichenbach, H. J. Antibiot. 1987, 40,
7.