Truncated borrelidin analogues: Synthesis by sequential cross metathesis/olefination for the southern fragment and biological evaluation

Article abstract of DOI:10.1039/c6ob01358a

The construction of novel borrelidin analogues is reported in which the northern fragment is truncated to a simple hydroxyundecanecarboxylate and the original cyclopentanecarboxylic acid in the southern fragment is replaced with different six-membered rings. The required precursors were prepared by cross metathesis of the appropriate carbocycle-based homoallylic alcohol with crotonaldehyde followed by HWE olefination of the resulting enal with bromocyanophosphonate. The key aldehyde for intramolecular cross coupling was accessible by oxidation of the hydroxy group of the linked undecanecarboxylate unit. Grignard mediated macrocyclization finally yielded the borrelidin related products. The investigation is complemented by SAR studies and quantum-chemical calculations.

Full text of DOI:10.1039/c6ob01358a

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Page 1 of 22
Organic & Biomolecular Chemistry
Truncated Borrelidin Analogues: Synthesis by Sequential Cross Metathesis/
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Olefination for the Southern Fragment and Biological Evaluation†DOI: 10.1039/C6OB01358A
Tülay Gündemir-Durmaz,a Fabian Schmid,a Yana El Baz,a Annette Häusser,a Carmen
Schneider,b Ursula Bilitewski,b Guntram Rauhut,c Delphine Garnier,a Angelika Baroa and
Sabine Laschat*a
The construction of novel borrelidin analogues is reported in which the northern fragment is
truncated to a simple hydroxyundecanecarboxylate and the original cyclopentanecarboxylic acid
in the southern fragment is replaced with different six-membered rings. The required precursors
were prepared by cross metathesis of the appropriate carbocycle-based homoallylic alcohol with
crotonaldehyde followed by HWE olefination of the resulting enal with bromocyanophospho-
nate. The key aldehyde for intramolecular cross coupling was accessible by oxidation of the
hydroxy group of linked undecanecarboxylate unit. Grignard mediated macrocyclization finally
yielded the borrelidin related products. The investigation is complemented by SAR studies and
quantum-chemical calculations.
a Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart,
Germany.
E-mail: sabine.laschat@oc.uni-stuttgart.de
b AG Compound Profiling and Screening, Helmholtz Zentrum für Infektionsforschung, Inhoffen-
str. 7, D-38124 Braunschweig, Germany
c Institut für Theoretische Chemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart,
Germany
†Electronic supplementary information (ESI) available: general procedures and syntheses of the
described derivatives, preliminary studies on olefination reactions, NMR studies on the stereo-
chemistry of the terminal double bond in bromocyanodiene (4En)dBr, quantum-chemical calcu-
lation of (27Ar) as well as 1H and 13C NMR spectra for all new compounds.
1

Organic & Biomolecular Chemistry
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Introduction
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DOI: 10.1039/C6OB01358A
Polyketide natural products consist of a huge variety of structurally diverse compounds originat-
ing from a modified fatty acid biosynthesis. They display a broad spectrum of biological activi-
ties relevant for medicinal chemistry and drug development.1 A prominent member of this
family is (–)-borrelidin (1) (Scheme 1), which was isolated by Berger et al. in 1949 from Strep-
tomyces rochei.2 Apart from its activity against borrelia, the major cause of lyme desease,2–4
a
broad biological profile has been discovered. Borrelidin (1) displays antiviral activity,5 inhibits
angiogenesis6 as well as several enzymes such as cyclin-dependent kinase Cdc28/Cln2 of
Saccharomyces cerevisiae7 and threonyl tRNA synthetase.8 In addition, anti-malaria activity
against chloroquine-resistant strains of Plasmodia falsiparum has been reported.9 The promising
biological activities have stimulated several synthetic endeavours3 and up to now five total
syntheses have been reported by the groups of Morken,10 Hanessian,11 Theodorakis,12 Omura13
and Minnaard14 complemented by synthetic studies.15 Hahn et al. synthesized complex inter-
mediates in order to probe the dehydratase from the borrelidin biosynthesis.16 By employing
precursor-directed biosynthesis Wilkinson et al. prepared several borrelidin analogues with
modified C-17 side chain and were able to separate anti-angiogenetic from cytotoxic acti-
vities.17,18 Omura's group reported that esterification of the free carboxylic acid with various
triazolyl-containing alcohols led to improved anti-malaria activity and reduced cytotoxicity.9
With borrelidin B containing an aminomethyl group in place of the nitrile function the need of
the CN group for threonyl tRNA synthetase inhibition was demonstrated.19 In a comparative
cytological study loss of biological activity for borrelidin congeners with amide functions at C-
12 and C-22 was observed.20 In all cases, however, the macrocycle of 1 was retained. These
results motivated us to study borrelidin analogues with modified macrocyclic skeleton regarding
their potential cytotoxic properties. Taking the concept of truncated natural products discussed
by Gademann et al.21 and Maier22 and successfully demonstrated by Nakata23 for kendomycin
analogues into account, we aimed to prepare the borrelidin related compounds 2 with different
2

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rings in the side chain at C-17 and the complex polyketide region truncated to a simple 11-
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DOI: 10.1039/C6OB01358A
hydroxyundecane chain (Scheme 1). The retrosynthesis based on disconnection of 2 into
northern (3) and southern fragment 4, which can be traced back to known 11-bromoundecanoic
acid and precursors 5. The latter require different routes starting from known compounds.
Scheme 1 Retrosynthetic approach to borrelidin analogues 2 with different rings at C-17 and
truncated northern fragment.
As key steps for the connection of northern and southern fragments 3 and 4 an esterification and
C–C coupling following Omura's strategy13 or, alternatively, Grignard coupling according to
Iqbal's method24a was intended. To access the cyanodienes 4 from precursors 5, the suitability of
cross metathesis versus olefination or Knoevenagel-type condensation was studied based on
seminal contributions by Iqbal.24b The results towards synthesis and biological investigation of
southern fragments 4 and borrelidin analogues 2 are reported below.
3

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Results and Discussion
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DOI: 10.1039/C6OB01358A
Synthesis of cyanodienes 4a–c
According to a procedure by Jay-Smith et al.25 1,2-benzenedimethanol 6 prepared from diethyl
phthalate by LiAlH4 reduction26 was mono-protected with PMBCl to yield 7 in 88%. Subse-
quent Dess-Martin oxidation afforded the known aldehyde 8 in 95% yield (Scheme 2). Whereas
Sakurai reaction of 8 with allyltrimethylsilane in the presence of various Lewis acids27 either
gave no conversion at all or led to complete decomposition of the starting material, treatment of
8 with the corresponding Grignard reagent provided the desired alcohol (5Ar)a in 89% yield.
TBS protection of (5Ar)a gave derivative (5Ar)b in 91% yield. As we aimed at a maximum
flexibility with respect to the coupling of fragments 3 and 4 (Scheme 1), we also introduced the
acetyl protecting group in (5Ar)a under standard acetylation conditions, which would allow a
Yamaguchi macrolactonization as the intramolecular step. Precursor (5Ar)c served as a model
acyl-protected substrate for the synthesis of the southern fragment.
Scheme 2 Synthesis of benzene-based homoallylic alcohols (5Ar)a–c.
Cyclohexene-derived homoallylic alcohols (5En)a–c were prepared following a similar strategy
(Scheme 3). Acidic hydrolysis of 4-cyclohexene-1,2-dicarboxylic anhydride 928 followed by
LiAlH4 reduction and monoselective PMB-protection gave alcohol 10 in 78% yield over three
steps. After Dess-Martin oxidation, the resulting aldehyde 11 was submitted to a Grignard reac-
tion to yield homoallylic alcohol (5En)a in 95% as a diastereomeric mixture (dr 79 : 21), which
was separated by HPLC for characterization. TBS-protection or acetylation of diastereomeric
(5En)a provided target compounds (5En)b and (5En)c in 98% and 96% yield, respectively.
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DOI: 10.1039/C6OB01358A
Scheme 3 Synthesis of cyclohexene-based homoallylic alcohols (5En)a–c which were further
used as diastereomeric mixtures.
Cyclohexane-derived homoallylic alcohol (5Cy)a was prepared from diol 13, which was ob-
tained from the known D-dimenthyl succinate 12 by a Yamamoto asymmetric carbocycliza-
tion16,29,30 followed by LiAlH4 reduction (Scheme 4).
Scheme 4 Synthesis of cyclohexane-based homoallylic alcohols (5Cy)a,b and (5Cy)a'.
Mono-PMB protection of 13 and subsequent Swern oxidation afforded aldehyde 14 in 91% over
both steps. Grignard reaction of 14 with allylmagnesium chloride led to an inseparable
diastereomeric mixture of homoallylic alcohols (5Cy)a, (5Cy)a' (dr 78 : 22) in 86%. However,
5

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when (5Cy)a, (5Cy)a' were treated with TBSCl under our usual reaction conditions, only dia-
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stereomeric silylether (5Cy)b was isolated in 75% while diastereomeric alcohoDlO(I:51C0.1y03)9a/'C6rOeB-01358A
mained unreacted and could be recovered in 22%. This diastereoselective kinetic resolution was
rather unexpected, although some examples of diastereoselective Si–O couplings existed, e.g.
employing silicon-stereogenic hydrosilanes and achiral Cu complexes.31,32 Presumably, the
reactivity of the OH group in diastereomer (5Cy)a' is diminished by hydrogen bonding between
OH and the PMB group.
With homoallylic alcohols 5 in hand we investigated the cross metathesis with crotonaldehyde
15 (Table 1), which was reported to give better yields than acrolein when using Grubbs II
catalyst.33
Table 1 Cross metathesis of homoallylic alcohol derivatives 5 with crotonalde-
hyde 15 to enals 16
Entrya
5
R1
Time (h) 16
Yieldb (%) dr
1
2
(5Ar)b
(5Ar)c
(5En)a
(5En)b
(5En)c
(5Cy)a'
(5Cy)b
TBS
Ac
H
TBS
Ac
H
20
18
19
20
18
18
20
(16Ar)b
35
89
88
92
84
(16Ar)c
(16En)a
(16En)b
(16En)c
(16Cy)a' 79
(16Cy)b 80
3
79:21
83:17
73:27
4c
5
6
7c
TBS
a Reaction conditions: 5 (1.0 equiv.), 15 (1.0 equiv.), cat. (5 mol%). b Isolated
yield. c Further addition of Grubbs II (5 mol%) after 18 h.
Treatment of aromatic TBS-protected cross metathesis (CM) precursor (5Ar)b with croton-
aldehyde 15 in the presence of second generation Grubbs catalyst (Grubbs II) (5 mol%) in
CH2Cl2 at 45°C provided 35% of the desired product (16Ar)b (entry 1). Cross metathesis of
cyclohexane (5Cy)b afforded 80% of enal (16Cy)b on addition of further Grubbs II catalyst
6

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Organic & Biomolecular Chemistry
after 18 h reaction time (entry 7). Under these conditions the corresponding cyclohexene deri-
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DOI: 10.1039/C6OB01358A
1
vative (5En)b gave 92% of diastereomeric CM product (16En)b (dr 5 : 1 by H NMR of the
CHO signal) (entry 4). As can be seen in Table 1, CM products (16En)a and (16Cy)a' with free
hydroxy group were isolated in high yields of 88% and 79%, respectively (entries 3, 6). This
observation is in good agreement with previous reports by Fuwa et al.34 and Lin and Davis35 that
free hydroxy groups have a beneficial influence on cross metathesis reactions due to hydrogen-
bonding of the OH group with the chlorine atom of the ruthenium carbene complex.36 Also the
acetylated precursors (5Ar)c and (5En)c underwent cross metathesis under similar conditions
providing the desired CM products (16Ar)c and (16En)c in comparable yields (entries 2, 5).
The synthesis of cyanodiene fragments 4 was studied using first cross metathesis enals
(16En)a,b (Scheme 5). Knoevenagel condensation of (16En)b with chloro- or bromoacetonitrile
17a,b37 to cyanodiene (4En)b, however, completely failed. An alternative strategy used a se-
quence of Wittig reaction/cross metathesis. While the Wittig olefination of enals (16En)a,b with
methyltriphenylphosphonium bromide and nBuLi proceeded uneventfully to the dienes (18En)a
and (18En)b in 31% and 54% yield, respectively, subsequent cross metathesis of (18En)b with
substituted methacrylonitrile 1938 gave no conversion to cyanodiene (20En)b.
Scheme 5 Preliminary studies on olefination reactions of (16En)a,b to give fragments (4En)a,b
(for details see ESI†).
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Therefore, we decided to follow Omura’s initial approach13 by utilizing electron-poor bromo-
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DOI: 10.1039/C6OB01358A
cyanophosphorane 2139 or cyanophosphonates 2240 and again enals (16En)a,b as benchmark
substrates for the olefination. While the reaction of unprotected derivative (16En)a with phos-
phorane 21 at room temperature in CH2Cl2 yielded the desired olefination product (4En)a in
38%, the corresponding TBS-protected (16En)b gave no conversion (Scheme 5). As afterwards
neither (16Ar) nor (16Cy) enals reacted under these conditions (ESI†), the Wittig reaction was
abandoned.
Next, the HWE olefination of enals 16 with -chloro- and -bromocyanophosphonate 22a,b,
respectively, was investigated. The results are summarized in Table 2. Deprotonation of -
chlorocyanophosphonate 22a with NaH in DMF at 0°C followed by addition of substrate
(16En)a with free OH group or the TBS-protected analogue (16En)b at 0°C provided the
respective cyanodienes (4En)aCl and (4En)bCl in 57% and 53% yield (entries 4, 8). In both
cases, however, reaction with the corresponding -bromocyanophosphonate 22b under similar
conditions did not lead to the olefination products (4En)aBr and (4En)bBr (entries 5, 9). We
surmised that -bromocyanophosphonate 22b underwent nucleophilic displacement of the
bromide by hydride rather than deprotonation of the acidic -hydrogen.
Following Omura's reaction conditions13 we finally succeeded in the preparation of both chloro-
and bromocyanodienes 4. For this purpose, LiCl and subsequently DBU (1.5 equiv. each) were
added to a solution of the respective substrate 16 and cyanophosphonate 22a or 22b in
acetonitrile at 0 °C (Table 2). In this manner bromocyanodiene (4En)bBr could be isolated in
48% (entry 11). Similar yields were obtained for the unprotected enal (4En)a irrespective of the
halide (entries 6, 7). The HWE olefination of cyclohexane-based substrates (16Cy)a',b resulted
in good yields for the bromocyanodienes (4Cy)a'Br (77%) and (4Cy)bBr (76%) (entries
15, 19), while the yields of the corresponding chloro compounds (4Cy)a'Cl and (4Cy)bCl
decreased to 43% and 27%, respectively (entries 14, 18).
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Organic & Biomolecular Chemistry
Table 2 HWE olefination of enals 16 with -halogenocyanophosphates 22 to cyanodienes 4
Entrya Enal
R1
22
Base
NaH
Solvent Time (h) Product
Yieldb (%) drc
1d
2
(16Ar)a
H
(16Ar)c Ac
(16Ar)c Ac
22b
22a
22b
22a
22b
22a
22b
THF
48
(4Ar)bBr
(4Ar)cCl
(4Ar)cBr
(4En)aCl
(4En)aBr
(4En)aCl
–
–
DBU MeCN 1.5
DBU MeCN 1.5
NaH
NaH
DBU MeCN
DBU MeCN
NaH
NaH
DBU MeCN
DBU MeCN
DBU MeCN 1.5
NaH
DBU MeCN
DBU MeCN
11
65
57
n.d.
n.d.
80:20
–
3
4
5
6
7
(16En)a
(16En)a
(16En)a
(16En)a
H
H
H
H
DMF
DMF
2.5
2.5
1
e
–
49
82:18
80:20
76:24
–
1
3
(4En)aBr 49
(4En)bCl 53
8
9
(16En)b TBS 22a
(16En)b TBS 22b
(16En)b TBS 22a
(16En)b TBS 22b
DMF
DMF
2.5
1
1
(4En)bBr
–
10
11
12
13
14
15
16
17
18
19
(4En)bCl 51
(4En)bBr 48
(4En)cBr 99
78:22
78:22
82:18
–
(16En)c Ac
22b
22a
22a
22b
(16Cy)a'
(16Cy)a'
(16Cy)a'
(16Cy)b TBS 22a
(16Cy)b TBS 22b
(16Cy)b TBS 22a
(16Cy)b TBS 22b
H
H
H
DMF
3
(4Cy)a'Cl
–
1
1
(4Cy)a'Cl 43
(4Cy)a'Br 77
NaH
NaH
DBU MeCN
DBU MeCN
DMF
DMF
2.5
2.5
1
(4Cy)bCl
(4Cy)bBr
(4Cy)bCl 27
(4Cy)bBr 76
–
–
–
–
74:26f
85:15f
1
a Reaction conditions: 16 (1.0 equiv.), 22 (2.0 equiv.), LiCl (1.5 equiv.), DBU (1.5 equiv.); 16 (1.0
equiv.), NaH, 22 (1.25 equiv. each). b Isolated yield. c dr refers to stereocentre C-1 at the homoallylic
ether position relative to the fixed cis or trans configuration in (4En) and (4Cy). d Temperature:
20°Cr.t. e No conversion, starting materials were reisolated. f E/Z ratio at C-6.
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This difference in yields between chloro- and bromocyanodiene became even larger when
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acetyl-protected enal (16Ar)c was olefinated to (4Ar)cCl (11%) and (4ArD)cOBI: 1r0.10(3695/C%6O)B01358A
(entries 2, 3). A similar trend of increasing yield was observed for the olefination of ace-
tylated (16En)c to (4En)cBr (99%) (entry 12).
Synthesis of borrelidin analogues 2
In order to optimize the reaction conditions for the envisioned cross coupling of fragments 3
and 4 we first studied the reaction of bromocyanodienes (4)Br with isobutyraldehyde 23 as a
model substrate for fragment 3 (Table 3).
Table 3 Intermolecular cross coupling of cyanodienes 4 with isobutyraldehyde 23 to
alcohols 24
Entrya Diene
Method Product
Yieldb (%) drc
(24Ar)c 18 66:34 –
25
Yieldb (%)
1
2
3
4
5
(4Ar)cBr
(4Ar)cBr
A
B
–
(24Ar)c 28
(24En)b 37
52:48 (25Ar)c 41
(4En)bBr B
(4En)cBr A
(4Cy)bBr B
83:17 –
67:33 –
–
–
(24En)c
8
(24Cy)b 48
89:11 (25Cy)b 28
a Reaction conditions: 4 (1.0 equiv.), 23 (1.0 equiv.), SmI2 (29 equiv.), DMPU (19
equiv.); iPrMgBr (1.1 equiv.). b Isolated yield. c Determined by integration in 1H NMR
spectra.
According to Omura's conditions,13b aryl-substituted acetoxycyanodiene (4Ar)cBr was treat-
ed with aldehyde 23 and a large excess of SmI2 (29 equiv.) in the presence of DMPU instead
10

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Organic & Biomolecular Chemistry
of HMPA in THF (method A). The secondary alcohol (24Ar)c could be isolated albeit in a
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low yield of 18% (dr 66 : 34) (entry 1). In contrast, treatment of (4Ar)cBr withDiOPI:r1M0.1g03B9/rC6iOnB01358A
THF according to Iqbal’s method,24 followed by addition of 23 (method B) slightly increased
the yield to 28% (dr 52 : 48), but alcohol (24Ar)c was accompanied by a byproduct (25Ar)c
being isolated in 41% as an E/Z mixture (11 : 89) (entry 2). Its presence indicated that the
initial attack of the isopropyl Grignard to fragment (4Ar)cBr and subsequent halogen metal
exchange had indeed taken place, but subsequent addition of the in situ formed Grignard
seemed to be too slow and thus, hydrolysis of the cyanodiene Grignard species occurred
during workup giving byproduct (25Ar)c. Under both Grignard and SmI2 conditions,
cyanodienes (4En)bBr and (4En)cBr reacted to alcohols (24En)b and (24En)c which were
isolated in low yields of 37% and 8%, respectively (entries 3, 4). The Grignard addition of
cyclohexyl-substituted fragment (4Cy)bBr gave alcohol (24Cy)b in 48% yield (dr 89 : 11)
together with 28% of the dehalogenated byproduct (25Cy)b (entry 5). The results in Table 3
revealed that intermolecular cross coupling could be achieved under both SmI2 and Grignard
mediated conditions, however, with less satisfactory yields for the SmI2 method. We thus
anticipated a poorer performance of SmI2 in the intramolecular cyclization as compared to the
Grignard reaction.
Keeping the obtained results in mind we continued with the synthesis of the macrocyclic
borrelidin analogues 2 as outlined in Scheme 6. Homoallylic alcohols (5Ar)a and (5En)a
were esterified under Yamaguchi conditions41 with northern fragment 3 prepared in 99% yield
by tetrahydropyranylation38b of 11-hydroxyundecanoic acid42 to yield the corresponding car-
boxylates (5Ar)d, (5En)d in 86% and 91%, respectively. Cross metathesis with croton-
aldehyde 15 in the presence of Grubbs II catalyst under the optimized conditions provided
enals (16Ar)d, (16En)d in 81% and 54% yield, respectively. The HWE reaction between the
latter and bromocyanophosphonate 22b gave the olefination products (4Ar)dBr and
(4En)dBr in 59% and 42%, respectively. The decreased yields might be caused by a lower
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solubility of the less polar precursors (16Ar)d, (16En)d in acetonitrile as compared to model
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DOI: 10.1039/C6OB01358A
compound (16Ar)c carrying a simple acetate protecting group. THP deprotection with PPTS
in EtOH proceeded uneventfully to afford alcohols (26Ar) and (26En) in 84% and 93% yield,
which were subsequently oxidized with TPAP under Ley conditions43 to yield aldehydes
(27Ar) and (27En) in 88% and 78%, respectively, the key intermediates for the intra-
molecular cross coupling. The intermolecular cross coupling conditions (Table 3) were trans-
ferred to the intramolecular coupling reaction.
Scheme 6 Synthesis of borrelidin analogues (2Ar) and (2En). For attempts to assign the
stereochemistry of the terminal C=C double bond in derivative (4En)dBr see ESI†.
The SmI2 mediated Reformatsky-type macrocyclization of (27Ar) under high dilution
according to Omura (method A) failed to give the desired macrocycle (2Ar). Only the open-
chain cyanodiene (25Ar)e (R1 = HO(CH2)10CO–, see also Table 3 and ESI†) resulting from
SmI2 induced reduction of the aldehyde moiety to the primary alcohol and simultaneous
reductive debromination was isolated in 27%. In contrast, the cross coupling of (27Ar) with
iPrMgBr in THF (method B) by Grignard reaction succeeded. The amount of iPrMgBr,
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however, was increased to 2 equivalents and the reaction time for halogen metal exchange
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prolonged to 1 h. After stirring at room temperature for 36 h followed by aqueousDwOIo: 1r0k.1u0p39,/Cth6OeB01358A
desired borrelidin analogue (2Ar) was isolated in 11% yield. In the case of cyclohexenyl-
derived compound (27En), only small amounts of the target macrocycle (2En) were detected
after 36 h reaction time under similar conditions. However, reaction time extension to 9 days
resulted in the macrocyclic target compound (2En) in 11% yield.
We assume that the northern fragment 3 might be responsible for the moderate yields of the
Grignard induced macrocyclization and the complete failure of SmI2 promoted intramolecular
reactions of aldehydes 27. In comparison, Omura obtained 60% of the desired macrocycle
with the 1,3,5,7-tetramethylated northern fragment in the case of parent borrelidin synthesis.13
These results might arise from the different conformations of the non-branched and the
methyl-branched fragment. In order to investigate this aspect in detail, we have performed
quantum-chemical calculations based on density functional theory (B3LYP/cc-pVDZ) for
(27Ar). Out of a large number of conformers we isolated 27A and 27B shown in Figure 1.
After correction for the zero-point vibrational energy, conformer 27A was found to be 2.6
kcal mol–1 lower in energy than conformer 27B, which constitutes a possible precursor to the
cyclization reaction. As slight distortions of the 27B structure led to conformer 27A, the
barrier between these two conformers must be very low. The opposite was found for the
methyl-branched system: distortions of the analogue of conformer 27B did not lead to the
analogue of conformer 27A, but the system was always trapped in the local minimum of the
structure of the 27B analogue. As a consequence, it is the occurrence of a multitude of
conformations (including those with a linear northern fragment), which are lower in energy
than the precursor 27B shown in Figure 1, and which are connected by low barriers, being
responsible for low yields and/or long reaction times.
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Fig. 1 Different conformations of (27Ar) (a, b). The conformer 27A is about 2.6 kcal mol–1
lower in energy than 27B (B3LYP/cc-pVDZ) (for further conformers see ESI†). For compari-
son the methyl-branched borrelidin analogue of 27B is depicted (c).
Biological studies of cyanodienes and borrelidin analogues 2
The ability of a set of cyanodiene derivatives 4, 24, 26, and borrelidin analogues (2Ar) and
(2En) to inhibit the proliferation of the L-929 mouse fibroblast cell line was examined using
the WST-1 cytotoxicity assay according to the protocol given by the manufacturer.44 The
results are summarized in Table 4.
As can be seen from the data in Table 4, the studied derivatives 4, 24, and 26 generally re-
vealed only moderate cytotoxicity against the L-929 mouse fibroblast cell line with IC50
values in the range of approximately 13–38 M (entries 4,5,7,8,11–13,15,17–20), which are
approximately 2 orders of magnitude higher than the IC50 value of borrelidin itself (entry 1).
14

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Organic & Biomolecular Chemistry
Table 4 Cytotoxic activities of cyanodienes 4, 24, 26 and borrelidin
analogues 2 against the L-929 mouse fibroblast cell line. For comparison
the corresponding value of borrelidin (1) was determined.
View Article Online
DOI: 10.1039/C6OB01358A
Entrya Compound IC50 (M)
borrelidin 1 0.49±0.19
Entrya Compound IC50 (M)
1
11
12
13
14
15
16
17
18
19
20
(4En)cBr
(4Cy)a'Cl
(4Cy)aBr
(4Cy)bCl
(4Cy)bBr
(24Ar)c
(24En)b
(24Cy)b
(26Ar)
27.1±1.5
12.9±2.7
16.9±3.4
>50
37.8±5.1
–
16.8±4.7
14.5±4.4
22.3±9.8
16.4±3.9
2
(2Ar)
>50
3
(2En)
>50
4
5
6
7
8
9
10
(4Ar)aBr
(4Ar)cCl
(4Ar)cBr
(4En)aCl
(4En)aBr
(4En)bCl
(4En)bBr
25.0±0.7
37.6±10.8
>50
23.3±7.9
20.5±9.6
–
–
(26En)
a IC50 values were calculated by fitting the concentration dependence of the
signals from the WST-1 cytotoxicity assay44 with the 4-parameter equation
and are given as mean ± S.D. of four replicates.
Cyclohexane-based fragment (4Cy)a'Cl with the free hydroxy group was the most active one
in this series (IC50 12.9±2.7 M). The presence of 11-hydroxyundecanecarboxylate in com-
pound (26Ar) seems to influence the cytotoxic activity when compared with acetyl-protected
(4Ar)cBr (entries 6, 19), as only (26Ar) displayed potency against the cell line (IC50 22.3±9.8
M). This effect was less pronounced for the corresponding cyclohexene-based derivatives
15

Organic & Biomolecular Chemistry
Page 16 of 22
(4En)cBr and (26En) (entries 11, 20). Also the activity of cyanodienes 24 appeared to depend
View Article Online
on the O-protecting group. Whereas acetyl-protected (24Ar)c was inactive,DOTI:B10S.1-0e3t9h/Ce6rOsB01358A
(24En)b and (24Cy)b revealed IC50 values between 15–17 M, respectively (entries 16–18).
Both borrelidin analogues (2Ar) and (2En) were inactive (entries 2, 3) showing the relevance
of the residue at C-17 for the biological activity of intact borrelidin. However, when only the
southern fragment is present, the cytotoxicities seem to be only slightly affected by the kind
of the six-membered ring system.
It should be noted that Sugawara et al. reported a significant decrease of cytotoxicity on the
human diploid embryonic cell line MRC-5 when the carboxylic acid at C-22 in borrelidin (1)
was replaced by a methylester.9a Furthermore, the cytotoxic activity was almost lost upon
acetylation of the 3-OH and 11-OH group of 1. Although comparison of these data should be
handled with great care and taking into consideration that the mode of action of these
analogues and the natural product might be different due to significant structural differences,
the results in Table 4 suggest that an unbiased halogenocyanodiene moiety apparently favors
cytotoxicity, whereas the macrocyclic ring deteriorates the activity (e.g. compare entries 2, 4,
19). The increase of biological activity of borrelidin (1) by one order of magnitude upon
hydrogenation of the C12–C15 diene moiety9a indicates that the contribution of the
cyanodiene unit to the biological mode of action as well as the role of the northern fragment
and the interplay between northern and southern fragment needs to be further studied.
Conclusion
We have prepared a dedicated library of cyanodienes employing a sequence of cross meta-
thesis and HWE olefination as key steps while alternative approaches met with little success.
Cyanodienes were designed as structural analogues of the southern fragment of borrelidin (1).
Attempts to cyclize the cyanodienes (27Ar) and (27En) to the corresponding macrocyclic
borrelidin analogues (2Ar) and (2En) by using Omura's SmI2 method were not successful.
However, this key transformation could be achieved by Grignard cross coupling providing the
16

Page 17 of 22
Organic & Biomolecular Chemistry
borrelidin analogues (2Ar) with aryl side chain at C17 and the corresponding analogue (2En)
View Article Online
with cyclohexenyl side chain at C17 in 6 steps starting from fragments (5Ar)a DaOnId: 10(.150E39n/C)6aO.B01358A
Quantum-chemical calculations were performed for (27Ar) to interpret the macrocyclization.
The calculation suggests that the northern fragment, i.e. the 11-hydroxyundecanecarboxylate,
makes the macrocyclization entropically less favorable due to a multitude of flexible con-
formations which are connected by low barriers. The opposite was found for the highly
methyl-branched fragment in the natural product 1. For the latter a helical conformation has
already been reported11,15a which seems to facilitate the macrocyclization. SAR studies
employing the L-929 mouse fibroblast cell line indicate a higher cytotoxicity for unbiased
cyanodienes 4, 24, 26 than for the truncated borrelidin derivatives 2. Future work must
demonstrate whether more rigified northern fragments are beneficial with regard to both
macrocycle formation and biological activity.
Acknowledgements
Generous financial support by the Dr. Eugen Ebert Stiftung (fellowship for T.G.-D.), the
Ministerium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg, the
Fonds der Chemischen Industrie and the GDCh (travel grant for T.G.-D.) is gratefully
acknowledged. The authors gratefully acknowledge the technical assistance of Yasmin
Wenzel (AG COPS, HZI) and the support of Dr. A. Raja (Dept. CBIO, HZI) with the
biological profiling of the compounds.
Notes and references
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