Exploring the Ring-Closing Metathesis for the Construction of the Solomonamide Macrocyclic Core: Identification of Bioactive Precursors

Article abstract of DOI:10.1021/acs.joc.7b02988

New synthetic strategies directed toward the novel cyclopeptides solomonamides have been explored utilizing an olefin metathesis as the key reaction. In the various strategies investigated, we worked on minimally oxidized systems, and the olefin metathesis reaction demonstrated efficiency and validity for the construction of the macrocyclic core. The described synthetic strategies toward the solomonamides are well suited for the subsequent access to the natural products and represent flexible and diversity-oriented routes that allow for the generation of a variety of analogues via oxidative transformations. In addition, preliminary biological evaluations of the generated solomonamide precursors revealed antitumor activity against various tumor cell lines.

Full text of DOI:10.1021/acs.joc.7b02988

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Exploring the Ring-Closing Metathesis for the Construction of the
Solomonamide Macrocyclic Core: Identification of Bioactive Precursors
Iván Cheng-Sánchez, Paloma Carrillo, Antonio Sánchez-Ruiz, Beatriz Martínez-Poveda,
Ana R. Quesada, Miguel Ángel Medina, Juan Manuel López-Romero, and Francisco Sarabia
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02988 • Publication Date (Web): 20 Apr 2018
Downloaded from http://pubs.acs.org on April 20, 2018
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Exploring the Ring-Closing Metathesis for the
Construction of the Solomonamide Macrocyclic Core:
Identification of Bioactive Precursors
[a]
[b]
[c]
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Iván Cheng-Sánchez, Paloma Carrillo, Antonio Sánchez-Ruiz, Beatriz
[
b]
[b]
[b]
Martínez-Poveda, Ana R. Quesada, Miguel A. Medina,
[
a]
[a]
Juan M. López-Romero and Francisco Sarabia
[
a]
Department of Organic Chemistry; Faculty of Sciences. University of Malaga. Campus de
Teatinos s/n. 29071. Malaga (SPAIN)
[
b]
Department of Biochemistry and Molecular Biology; Faculty of Sciences. University of
Malaga. Campus de Teatinos s/n. 29071. Malaga (SPAIN)
[
c]
Organic Chemistry Section; Faculty of Pharmacy. University of Castilla-La Mancha. Avda
Dr. José María Sánchez Ibáñez s/n. 02008. Albacete (SPAIN)
Telephone: 34-952 134258. Fax: 34-952 131941
Email: frsarabia@uma.es
HL60
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Abstract: New synthetic strategies directed towards the novel cyclopeptides solomonamides
have been explored utilizing an olefin metathesis as the key reaction. In the various strategies
investigated, we worked on minimally oxidized systems and the olefin metathesis reaction
demonstrated efficiency and validity for the construction of the macrocyclic core. The described
synthetic strategies towards the solomonamides are well suited for the subsequent access to the
natural products and represent flexible and diversity-oriented routes that allow for the
generation of a variety of analogues via oxidative transformations. In addition, preliminary
biological evaluations of the generated solomonamide precursors revealed anti-tumor activity
against various tumor cell lines.
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1. INTRODUCTION
Recently isolated from the marine sponge Theonella swinhoei, the solomonamides A (1) and B
2) (Figure 1) are natural products with interesting and promising biological properties.1
(
Structurally characterized by unprecedented cyclopeptidic-type frameworks, these natural
products have become potential leads for drug discovery and join a broad and impressive family
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of other bioactive natural products provided by this marine sponge. An exhaustive
spectroscopic analysis of both compounds has resulted in the elucidation of their intricate cyclic
structures, revealing the presence of three conventional amino acids (D-Ala, Gly and L-Ser)
and an unprecedented 4-amino(2’-amino-4’-hydroxyphenyl)-3,5-dihydroxy-2-methyl-6-
oxohexanoic acid (ADMOA) and its corresponding 5-deoxy derivative (AHMOA) for
solomonamides A and B, respectively. Assignment of the absolute configurations of ADMOA
and AHMOA required additional studies, involving a combination of spectroscopic and
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theoretical studies (QM J based analysis and DFT J/ C calculations), which resulted in the
proposal of the depicted absolute configurations as the most likely. Biologically, solomonamide
A (1) displayed potent anti-inflammatory activity, causing a significant 60% reduction of
inflammation in an animal model of edema at 100 μg/Kg. Unfortunately, the extreme scarcity
of the solomonamides has precluded a thorough biological evaluation. In fact, the anti-
inflammatory activity of solomonamide B (2) was not evaluated due to limited amounts. The
unique and unprecedented structures of the solomonamides, together with their intriguing
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biological properties, have generated intense synthetic activity. For example, the Reddy group
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has recently reported a total synthesis of a deoxy analogue of solomonamide B together with
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an array of simple unfunctionalized analogues, culminating with a total synthesis of the natural
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solomonamide B, which has led to a revision of the initially proposed structure as will be
detailed later.
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Figure 1. Originally Assigned Structures of the Solomonamides
Our ongoing interest in the discovery and development of new potential leads based on
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cyclopeptidic- and cyclodepsipeptidaic-type compounds, prompted us to initiate a research
program directed toward the total synthesis of this novel and unexplored class of cyclopeptides.
With the aim of establishing a flexible and divergent synthetic strategy capable of providing
not only the natural products, but also provide an entry into a plethora of analogues for
biological studies, we sought to explore the ring-closing metathesis (RCM) reaction as the key
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step for construction of the macrocycle. This cyclisation step would be followed by an
oxidation phase, which would incorporate the functional groups needed to reach the final
oxidation stage found in the natural products. From a strategic perspective, we considered that
the construction of the macrocyclic core at the 4,5-bond would be capable of providing rapid
access not only to the final products, but also to analogues from late stage intermediates,
allowing for the facile entry into numerous scaffolds. Furthermore, it is worthy to note that this
synthetic strategy utilizes simple starting materials, avoiding the construction of the complex
ADMOA residue, which can be constructed in the later stages of the synthesis through an
epoxidation of the olefins 3-6, followed by an oxirane-ring opening process to introduce the
amine group. Accordingly, as detailed in Scheme 1, our delineated strategy in retrosynthetic
terms begins with the straightforward amide disconnection of the L-serine residue, followed by
the removal of the functional groups, which would be introduced by means of oxidative
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manipulations (oxidation phase) of the resulting metathesis products. In this way, the synthetic
4,5
strategy would render the corresponding macrocyclic alkenes represented by the cis-
derivatives 3-6, which would possess or not various functionalities at the benzylic position. All
these macrocyclic compounds, in turn, could be obtained from the corresponding acyclic
precursors 7-10 via a ring-closing metathesis process. Finally, the preparation of such
precursors would be achieved by simple peptidic-like assembly between the amine derivatives
of the corresponding Boc derivatives 11-12 and the olefinic acids 13 or 14 (Scheme 1, part A).
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Preliminary results in this synthetic direction have been recently published and support the
viability of this approach. In this manuscript we wish to report a full account of all the synthetic
studies carried out in our laboratories which have been initiated upon the basis of the previous
retrosynthetic scheme. In addition, during the execution of this synthetic work, Reddy et al
published the total synthesis of solomonamide B, based on an elegant intramolecular Heck
reaction, which provided the advanced solomonamide precursor 15. More importantly, the total
synthesis of solomonamide B led to the revision of the initially proposed structure for 2, with
the correction of the configurations at C-3 and at C-4 positions to the (3S, 4S)-isomer
(
compound 16) instead of the proposed (3R, 4R) for solomonamide B (2) (Scheme 1, part B).7
As the present synthetic studies were initiated prior to the Reddy publication, we targeted the
initially proposed structures for the solomonamides.
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Scheme 1. Retrosynthetic Analysis for the Solomonamides (A) and Synthetic Work by Reddy
(B)
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2. RESULTS AND DISCUSSION
.1. Ring-Closing Metathesis at the C4-C5 Bond. Encouraged by the appealing features of
2
this synthetic strategy, as mentioned above, we initiated the synthetic route. Our initial forays
toward the solomonamide structures were conducted to demonstrate the viability of the olefin
metathesis approach, based on the disconnection of the 4,5-positions bond, using the model
compounds 24 and 25 (Scheme 2). To this end, the readily accessible dipeptides 24 and 25 were
prepared from 2-bromo aniline 17 and the iodonitrobenzene derivative 18,11 respectively,
according to the synthetic sequence depicted in Scheme 2, entailing a Stille reaction for
compound 19, and a sequential Stille reaction/reduction, for compound 20. Couplings of the
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resulting anilines 19 and 20 with dipeptide 21 furnished the corresponding dipeptides 11 and
22, which, after Boc deprotection, were coupled with commercial acid 23 to yield the targeted
ring-closing metathesis precursors 24 and 25. Thus, 24 and 25 were treated with 10 mol% of
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Hoveyda-Grubbs 2 generation (HG-II) catalyst in refluxing dichloromethane in the presence
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of p-benzoquinone to obtain the expected macrocycles 26 and 27 in excellent 75 and 79%
yields, respectively, as the sole products of the reaction. The newly formed double bonds (Δ4,5)
of 26 and 27 were determined in both cases to be exclusively in the E-configuration, as
evidenced by a coupling constant J of 15.9 Hz, therefore revising our previous assignment,
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which was incorrect (Scheme 2).
Despite this stereochemical outcome, and with the possibility in mind that the structural pattern
of the acyclic precursor could influence the double bond geometry and switch in favour to the
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desired Z-isomer, we continued with the assembly of compounds 11 and 14 in a manner
similar to as described before for 24, providing 8 in a 74% overall yield. Upon exposure of 8 to
HG-II catalyst, the expected macrocycle 4 was not obtained, instead recovering starting
material, together with a significant degree of decomposition. Attributing steric factors to this
failed cyclisation, the silyl protecting group was removed by treatment of 8 with HFpyr to give
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allylic alcohol 7. Various reports in the literature, describing metathesis reactions involving
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allylic alcohols, indicate that these structural systems may favour the closing process, although
other studies point out that these systems result in detrimental effects for the metathesis
reaction.16 Nonetheless, when allylic alcohol 7 was treated with the HG-II catalyst in
dichloromethane at 40 ºC in the presence of p-benzoquinone, the E-olefin 28 was obtained
exclusively in a gratifying 71% yield, with no formation of the required Z-isomer 3, as revealed
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by the H NMR spectra of the crude reaction mixture (Scheme 3).
Scheme 2. Towards the Total Synthesis of Solomonamides: RCM of Model Compounds 24
and 25
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Scheme 3. Towards the Total Synthesis of Solomonamides: Synthesis of Macrocycle 28
In parallel to these preliminary works, we accomplished related synthetic studies extended to
more functionalised precursors that would provide for a shortened the path towards the
completion of the synthesis of these natural substances. In this direction, we pursued the
preparation of the macrocycles 5 and 6 (See scheme 1) as potential advanced precursors. In
order to rapidly inspect the validity of this approach, we initially worked with a model system
represented by the deoxy aromatic derivatives. Thus, the readily accessible allylic alcohol 2917
was transformed into the aniline 30 by treatment with Zn/NH Cl. Following the delineated
4
synthesis for previous compounds, 30 was coupled with dipeptide 21 to yield the coupled
product 31 as a 1:1 mixture of diastereoisomers. The assembly of 31 with acid 23 was preceded
by Boc deprotection under conventional acidic conditions, followed by amide coupling assisted
by HATU. Disappointingly, the expected coupling product 32 was not detected, instead the
derivative 33 was obtained as a result of a cationic rearrangement of the labile allylic alcohol
3
1, which probably occurred during the Boc deprotection step under the acidic conditions used
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(Scheme 4). Different attempts for removal the Boc group under mild acidic (TMSCl, TMSOTf,
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NaO Bu) resulted similarly unfruitful with the formation of rearranged byproducts, recovery
of starting material or degradation, respectively. As a consequence of these results, we
considered a direct coupling between aniline 30 and the peptidic fragment 36, which was
efficiently prepared by utilising solid phase peptide synthesis (SPPS) as described in Scheme
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. Thus, the coupling of aniline 30 and acid 36 provided the desired diolefinic precursor 32 in
a 77% yield. Finally, the ring-closing metathesis of 32 under the same conditions as in previous
cases, afforded the macrocyclic derivative 38 in a 73% yield as the E-isomer, supported by the
J4,5 coupling constant (14.6 Hz), and as a 1:1 mixture of diastereoisomers (Scheme 4).
Interestingly, the protected precursor 37, prepared by silylation of 32 with TBSOTf, did not
provide the corresponding macrocylic derivative when it was treated with the HG-II catalyst
under the same conditions used for 32, indicating that steric factors may be responsible for the
failed ring closure.
The extension of this synthetic scheme to the aromatic system contained in the natural products
1
9
was initiated with the aldehyde 40 prepared from commercially available 39. Subsequent
treatment of aldehyde 40 with magnesium vinylbromide provided allylic alcohol 41, which
required the reduction of the nitro group to the corresponding amine to give 42. This seemingly
simple operation however proved more problematic than expected. For example, treatment of
41 with Zn/NH Cl did not provide the expected aniline 42, instead a complex mixture of
4
unidentifiable degradation products were obtained. We surmised that the benzyloxy group
installed in the aromatic ring was playing a crucial role in the reactivity of the benzylic alcohol,
making it especially sensitive even under the weak acidic conditions of the reduction reaction.
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Scheme 4. Towards the Total Synthesis of the Solomonamides via Diolefin 32
To circumvent this rather disappointing outcome, we decided to protect the hydroxyl group as
the silyl ether 43. However, the Zn-mediated reduction of the nitro group did not provide the
expected aniline 44, instead the result again was the formation of a complex mixture of
degradation products. Other reduction methods, including LiAlH or Ni-Raney, were attempted
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but they also were unsucessful. Finally, the more robust BOM (PhCH
2
OCH -) protecting group
2
proved to be the solution for this problematic reduction, as the BOM derivative 45 provided the
expected aniline 46 when treated with Zn/NH Cl in a reasonable and reproducible 79% yield.
4
The linkage of aniline 46 and dipeptide 36 was then performed as described above for 32 to
obtain compound 47, which was subjected to the ring-closing metathesis to provide macrocycle
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8 in an excellent 84% yield as a 1:1 mixture of diastereoisomers and the E-olefin as the only
detectable double bond isomer. It is intriguing that this ring-closing metathesis process
proceeded in such good yield, despite the presence of a protecting group at the hydroxyl group
of the diolefinic precursor (Scheme 5).
Scheme 5. Towards the Total Synthesis of the Solomonamides via Diolefin 47
The implementation of this synthetic scheme onto the more functionalised system found in the
natural products was then confronted by the assembly of acid 49, prepared by SPPS (See
Experimental Part) and 46 under conventional conditions employed in this work
(HATU/DIPEA). However, no desired product 50 was obtained despite attempts with an array
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of coupling reagents such as BOP, PyBOP, DIC/HOBt or DIC/HOAt. Reasoning that steric
hindrances around the acid 49 could explain this failure, we attempted the synthesis of the
advanced precursor 50 by sequential couplings of aniline 46 with the amino acid derivatives 34
and 35 and the less sterically encumbered acid 14. Unfortunately, the last coupling with acid
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4 did not provide the expected product 50, instead a complex mixture of HATU derivatives
was obtained from the starting acid. Intrigued by these failed reactions, we turned our attention
to the simple aniline 30, in which the hydroxyl group at C6 position was free, in order to
determine the reasons of the serious hurdles found during this synthetic course. To this end,
aniline 30 and acid 49 were coupled by treatment with HATU/DIPEA and the corresponding
coupling product 51 was obtained, albeit in a low 36% yield, with the unexpected cleavage of
the silyl protecting group. Several attempts of this reaction revealed its lack of reproducibility,
confirming the difficulties of such a coupling. Although obtained in low yield, compound 51
was in hand and we were in position to investigate the ring-closing metathesis reaction for this
more complex system. The reaction, undertaken under similar conditions as in previous cases,
did not give the expected macrocycle 52, thus demonstrating the unsuitability of these substrates
in the construction of the macrocylic core of the solomonamides via RCM. The reason for
failure is most likely due to the sensitivity of the reaction to steric factors arising from the
presence of substituents at both allylic and benzylic positions (scheme 6).
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Scheme 6. Exploration of the RCM in More Complex Systems
In yet another attempt to access the macrocyclic solomonamide precursors functionalised at the
benzylic position, we considered the incorporation of a ketone at this position, since this is the
functional group present in the natural products. In addition, the generation of the ,-
unsaturated ketone system, after the macrocyclisation process, would allow for the rapid and
facile access to an epoxide via oxidation of the double bond. Notably, we were skeptical about
the success of the metathesis reaction in such a system, represented by compound 53, given that
the olefin is unactivated as it is conjugated (,-unsaturated phenylketone). Despite this
concern, we opted to experimentally verify the chemical reactivity of 53 under the action of the
Hoveyda-Grubbs and related catalysts.
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To this aim, we prepared ketone 53 by oxidation of the alcohol 32 with MnO and then,
2
nd
subjected to the catalytic action of the Hoveyda-Grubbs 2 generation, keeping in mind the
difficulty of this reaction due to the inactivated nature of the double bond of the ,-unsaturated
ketone system. Indeed, this reaction did not provide any desired product, even when the reaction
was forced using drastic conditions (ex. toluene at 60 ºC and 100 ºC), instead providing
recovered starting material in all cases. In another attempt to obtain cyclic ketone 54, cyclic
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alcohol 38 was treated with MnO
2
but the result was similarly unsuccessful, with only starting
material recovered and no detection of the coveted ketone 54 (Scheme 7).
Scheme 7. Exploration of the RCM of the Diolefinic ketone 53
4,5
Although a Z-geometry for the  double bond was proposed to provide access to the syn-1,2-
difunctionalized system present at these positions in the natural products, the fact that we
obtained instead the E-olefinic macrocycles in all cases represented an additional difficulty in
this study to obtain the final targets. Nonetheless, this synthetic strategy was not discarded at
this point, as synthetic methods are available to provide the required syn isomer from a trans
2
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double bond. Consequently, we decided to continue with the present synthetic approach and
the next step was to evaluate the feasibility of such compounds to provide more oxidized
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derivatives through an oxidation phase that would give access to the final products. In this
sense, we studied the oxidation of compounds 27, 28 and 48 as representative macrocyclic
precursors of the solomonamides. Thus, when 27 or 28 were subjected to the oxidative action
of m-CPBA, we found, to our dismay, that these reactions did not yield the expected epoxides
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5 or 56. Instead, starting materials were recovered in both cases. In light of these discouraging
results, we attempted the epoxidation utilizing the dioxirane derived from
2
1
trifluoromethylketone, however the result was similarly frustrating, with no formation of any
desired oxidation products. Other oxidative reactions were screened, such as a dihydroxylation
reaction mediated by OsO , and electrophilic additions, mediated by the actions of bromine or
4
NBS, but these did not provide favorable results, not detecting formation of any of the possible
oxidation products. The poor solubility observed for these cyclic compounds in common
organic solvents could explain the lack of reactivity found for them towards the oxidative
reagents. However, the more soluble derivative 48 also proved to be unreactive when it was
subjected to the epoxidation reagents (m-CPBAand dioxirane species), resulting in the recovery
of starting material and no detection of epoxide 57 (Scheme 8). Theoretical studies are currently
in progress in order to justify the lack of reactivity exerted by these macrocyclic compounds.
Thus, initial results from DFT calculations carried out in solution showed that the HOMO
orbital of the molecules is centered around the intracyclic phenyl ring with a low electronic
22
density around the alkene moiety. This result suggests that the main reactive point for the
oxidant should not be the alkene, as it could be expected, but the aromatic ring. At this stage,
no further work was required to recognize that oxidation of the previous solomonamide
precursors was extremely hard to accomplish. Therefore, a new approach was considered.
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Scheme 8. Attempts of Oxidation of the RCM Products
2
.2. Ring-Closing Metathesis at the C5-C6 Bond. Given the results obtained during the
synthetic studies towards the solomonamides utilizing the macrocyclic construction at the C4-
C5 bond, we turned our attention to the construction of the macrocyclic core of the
solomonamides at the C5-C6 bond. In relation to the synthetic strategy explored in the previous
section, the removal of the functional groups, which should be accessible during an oxidation
phase, would lead to the relatively simple precursor 58. Whereas the styryl double bond could
be transformed into a ketone or an -hydroxy ketone via Wacker or dihydroxylation oxidations
for solomonamides A and B, respectively, the introduction of the required amine group at the
C4 position was initially planned via a C-H activation. The syntheses of the key precursor 58
would be performed by a RCM of the acyclic derivative 59, whose synthesis would be achieved
by simple peptidic-like assemblies between the corresponding amine and olefinic acid. As in
the previous synthetic exploration, we preferred to initiate this study with the model compound
63 to test the viability of the new synthetic proposal. The preparation of this RCM precursor
23
was successfully achieved from the simple aniline 60 by sequential couplings with dipeptide
2
1 and hydroxy acid 6210,24 with HATU. With compound 63 in hand, we proceeded with the
olefin metathesis reaction by use of the HG-II catalyst in refluxing dichloromethane. However,
it was with much disappointment, that this reaction failed to afford any macrocyclic product,
leading instead to decomposition and/or polymerization, together with the recovery of some
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starting material (12%). Additional attempts that included more forcing conditions (toluene at
st
nd
st
6
5ºC or 100 ºC) and other catalysts (Grubbs 1 and 2 generations, Hoveyda-Grubbs 1
generation) were thwarted, with no detection of the desired macrocyclic product 64 (Scheme
9).
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Scheme 9. Second Approach to the Solomonamides via RCM: The C5-C6 Disconnection
Despite these discouraging results, we decided to press forward with the synthetic strategy by
exploring the ring closing metathesis in the real system represented by the product 59. We
reasoned that in the case of the metathesis precursor 59, the electronic effect of the benzyloxy
group at the para position with respect to the styryl double bond could exert a favourable effect
in terms of reactivity for the unreactive olefin. In order to confirm this hypothesis, we prepared
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the styryl derivative 67 from compound 65 in a very good overall yield. We then proceeded to
test the construction of the macrocyclic ring of the solomonamide model system 70. To this
end, compound 67 was coupled with commercial acid 68 to obtain the acyclic precursor 69 in
a 74% yield. Having prepared compound 69, we were primed to test our hypothesis regarding
the favourable electronic donating effect of the benzyloxy group on the ring-closing metathesis
reaction. Indeed, we were gratified to discover that cyclic compound 70 was obtained in a 43%
yield and, exclusively, as the E-isomer, when the acyclic precursor 69 was exposed to the HG-
II catalyst in refluxing dichloromethane in the presence of 1,4-benzoquinone (Scheme 10). In
light of this encouraging result, the ring-closing metathesis was extended to the advanced
intermediate 59, which was efficiently prepared from the Boc derivative 67 and acid 62 in 86%
overall yield, according to the previously described fragment coupling protocol. To our dismay,
treatment of 59 with the HG-II catalyst, under the same conditions previously employed in
earlier cases, did not provide the coveted solomonamide precursor 58, providing instead
degradation products and recovered starting material. A possible explanation for the failure of
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the metathesis reaction is the formation of either the five-membered ring intermediate A, by
chelation of the ruthenium carbenoid with the hydroxyl group, or the seven-membered ring
2
6
chelate intermediate B, in which sequestration of the ruthenium carbenoid species occurred
through a hydrogen bond of the hydroxyl group with the chlorine atom, which could explain
the inactivation of the catalyst for the RCM (Scheme 10).
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Scheme 10. RCM of the Acyclic Precursors 59 and 69
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As support for this mechanistic hypothesis, we decided to protect the hydroxyl group to avoid
the formation of the proposed unproductive intermediates. Therefore, the TBS derivative 71
was prepared in a good yield by treatment of 59 with TBSOTf in the presence of 2,6-lutidine.
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The resulting silyl ether 71 was then subjected to the exposure of the HG-II catalyst and, to our
delight, the macrocyclic product 72 was obtained in a reasonable 47% yield. The completion of
the synthesis of the natural products should require an eventual removal of the TBS protecting
group. With this future objective in mind, we proceeded with the desilylation reaction by
treatment of 72 with HFpyr. Surprisingly, the expected hydroxyl derivative 58 was not
obtained, instead recovered starting material. Other fluoride-based reagents (TBAF, TBAF-
AcOH) afforded the same frustrating result. Therefore, we decided to replace the TBS group
with a more labile protecting group, choosing TES as a suitable alternative. Then, compound
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3 was prepared by reaction of 59 with TESOTf/2,6-lutidine and its suitability as a viable
substrate for the preparation of 58 was evaluated. Gratifyingly, the RCM reaction of 73
provided 74 in a similar yield as for 72 (53%) and its desilylation, by treatment with TBAF,
yielded the desired macrocyclic 58 in 85% yield (Scheme 11).
Scheme 11. RCM of the Acyclic Precursors 71 and 73: Synthesis of 58
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2.3. Ring-Closing Metathesis at the C3-C4 Bond. Prior to the completion of the synthetic
study towards the natural solomonamides, for which meaningful quantities of 58 or precursor
7
4 were required, we opted to complete the exploration of the RCM reaction by exploring the
C6-C7 bond as another disconnection point to construct the macrocyclic system of the
solomonamides. This new disconnection strategy would be based on the retrosynthetic analysis
depicted in Scheme 12. Having established the viability of the RCM strategy for the
construction of the macrocylic core of the solomonamides at two different sites, the C5-C6 and
the C4-C5 bonds, we decided to extend the RCM-based strategy to the C3-C4 position, scanning
all the options along the six-carbons chain contained in the ADMOA fragment. In this new
scenario, the removal of the functional groups at the C3 and C4 positions revealed the
compound 75 as a potential precursor for solomonamide B, which could be obtained from the
acyclic derivative 76 via RCM. In the synthetic direction, aldehyde 40 was transformed into the
amine 79 without problems according to the synthetic sequence described in Scheme 12. Our
experience gathered during the synthetic campaign led us to choose BOM as the most suitable
protecting group for the benzylic alcohol and to introduce sequentially the two amino acids (D-
Ala and Gly) through the Fmoc derivatives 34 and 35 to obtain compound 81. Prior to the
preparation of the subtarget RCM precursor 76, we considered the use of the commercially
available acid 82 to test the possibilities of this new strategy. Having prepared model compound
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3, treatment with HG-II catalyst under similar reaction conditions as previously used, did not
provide the desired result with the no formation of the macrocyclic compound 84. In fact, a
rational explanation for this result could be the formation of a stable 5-membered ring chelate
between the ruthenium carbene and the carbonyl group of the amide (intermediate A), which
renders the metallacyclobutane intermediate unreactive toward the retro [2 + 2] cycloaddition28
(Scheme 12). At this stage, we decided to terminate this synthetic approach, taking into account
29
that this strategy could be reconsidered in the future by use of Lewis acids, which could be
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investigated as useful additives to promote the ring closure as has been demonstrated in cross-
metathesis reactions bearing similar structural motifs.
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Scheme 12. Third Approach to the Solomonamides via RCM: The C3-C4 Disconnection
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2.4. Biological Activity of Solomonamide Precursors.
At this stage of the synthetic work, with an efficient route defined to obtain the final natural
products, we were intrigued with the biological properties of the synthesized products,
recognizing that the macrocyclic peptides represented unprecedented and novel molecular
architectures of biological interest. For this reason, we decided to explore and investigate their
biological activities. To this aim, we performed preliminary biological evaluations, which
consisted of the measurement of the antitumor properties of selected compounds against a panel
of various tumor cell lines. The chosen solomonamide derivatives for this study were 26, 27,
0
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8, 38, 70, 74 and 58 as representative compounds of the different scaffolds generated during
the synthetic work. In addition, compound 85, obtained from 27 by treatment with Na/NH (See
3
Figure 2), was included for this study to evaluate the effect of the protecting group upon
biological activity.
Figure 2. Synthetic Solomonamide Precursors Submitted to Biological Evaluations
As a first attempt to characterize the biological activities of the synthetic solomonamide
analogues, the cytotoxicity profile of the aforementioned compounds was examined using nine
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different cancer cell lines (see Table 1), as well as a primary culture of non transformed bovine
30
aorta endothelial (BAEC) cells. The results of these biological evaluations, summarized in
Table 1, clearly revealed a relevant cytotoxic activity for only one compound, 58, which
displayed the best values of inhibition of the series in the low M range, against all the tumor
cell lines. In addition, 58 was also cytotoxic against the endothelial cells line (BAEC), which
may indicate a putative antiangiogenic effect of this compound.30 In contrast, the other
compounds were practically devoid of antitumor activity except compounds 28 and 70, which
displayed cytotoxicity, particularly against the HL60 tumor cell line albeit moderately. From
these results, a highlight is the notable enhancement of activity observed upon introduction of
functional groups in the polyketide fragment contained in these compounds, as concluded when
biological activities of 70 and 58 are compared, or 27 and 28 for the case of the HL60 cell line.
In addition, the isomeric derivative of 58, compound 28, displayed similar antiproliferative
activity as 58 against HL60, but, in contrast, was inactive against the other cell lines, including
BAEC, revealing the importance of the position of the double bond of the molecule upon
biological activities. This structural effect is also reflected when the biological activities of the
isomeric compounds 27 and 70 are compared. In an attempt to make sense of this result,
molecular modelling studies were conducted for compounds 27, 28, 70 and 58 to obtain the
corresponding minimized structures (Figure 3).31 The resulting models revealed a certain
structural distorsion when 27 and 70, on one side, and 28 and 58, on the other, are compared,
respectively. Thus, whereas 70 and 58 are almost flat, 27 and 28 displayed considerable
twisting. These differences in the resulting minimized molecular structures may explain the
differences observed for the biological activities found for these compounds. Additionally, the
overall shape of 27 is very similar to 28, and the same is evident also for 70 and 58. In this case,
despite these conformational similarities, it is clear that the functional groups incorporated in
1
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8 and 58, in particular the hydroxyl group, may exert a key biological interaction through a
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hydrogen bonding interaction with an acceptor-type residue located at the active site of the
biological targets. These interactions may justify the greater activities displayed by 28 and 58
compared with 27 and 70, respectively.
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Table 1. In vitro Antitumor Activities of Solomonamide Precursors against Various Tumor Cell
a
Lines and BAEC (IC50, M)
Compound
HL60b
KU812Fc
U937d
HT-
MDA-
MB-231
> 100
U87MGg
HepG2h
HT-29i
U2OSj
BAECk
e
f
1
080
2
6
> 50
> 50
> 50
> 50
n.d.
> 50
n.d.
> 100
> 100
> 100
> 50
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
27
> 100
> 100
> 50
43,8  1,2
> 100
8
2
3
5
8
8
> 50
> 50
> 100
> 100
> 100
> 100
17,2  6,6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
> 100
> 50
> 50
> 50
> 100
> 100
> 100
> 100
16,5 
> 100
> 100
> 100
> 100
> 100
70
20,5  5.2
> 50
> 50
31,1  2,8
> 100
> 100
> 100
> 100
69,6 12,5
7
5
4
8
> 50
> 50
> 50
> 100
> 100
> 100
> 100
> 100
> 100
14,7  4,4
14,0  3.05
7,02  2.32
16,3  2.9
34,8  5,1
18,9  1,5
13,3  0,6
12,9  3,1
18,1  2,2
1,01
[
a] In vitro cytotoxicities were determined according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction
assay as detailed in experimental part. The IC50 values were obtained from semilogarithmic dose-response plots as the concentration of
compound yielding a 50% of cell survival. [b] HL60: Human promyelocytic leukemia. [c] KU812F: Chronic Myelogenous leukemia. [d] U937:
Histiocytic lymphoma. [e] HT1080: Human fibrosarcoma. [f] MDA-MB-231: Human breast adenocarcinoma. [g] U87MG: Gioblastoma. [h]
HepG2: Hepatocellular carcinoma. [i] HT-29: Colorectal adenocarcinoma. [j] U2OS: Osteosarcoma. [k] BAEC: Non transformed bovine
aortic endothelial cells.
Figure 3. Computer-generated Minimum-Energy Conformations for Compounds 27, 28, 58
and 70
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3. CONCLUSIONS
In conclusion, an extensive synthetic exploration directed towards the solomonamides
was conducted based on a ring-closing metathesis as the key reaction for the rapid and
efficient access to their macrocyclic cores. The result of this synthetic study was the
establishment of an efficient ring closure process which proceeded in high yields and
complete stereoselectivity. During the course of these efforts, unexpected hurdles arose
along the way, mainly with: 1) the reactivity of the hydroxyl group at the benzylic
position (compounds 31 and 41); 2) the reactivity of diolefins containing allylic and
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homoallylic alcohols (compounds
unsaturated carbonyl system (case of compound 83) toward the ruthenium catalysts, and
) the reactivity of macrocyclic olefins (compounds 27 28 and 48) toward oxidative
7, 8, 32, 47, 59, 63, 71 and 73) or containing a ,-
3
,
reagents. Although many of these synthetic problems were overcome, others remained
elusive and represent synthetic challenges for future works. In relation to the ring-closing
metathesis reactions, many of the described findings support the observations and results
reported by other authors for this reaction on this class of structural systems. More
importantly, not only did we explore the scope and limitations of the ring-closing
metathesis in the synthesis of the macrocyclic core of the solomonamides, but we also
identified several structurally related solomonamide precursors possessing significant
cytotoxicities against various tumor cell lines, including endothelial cells, in the low M
range. These preliminary biological evaluations of relatively simple compounds, devoid
of the functional groups present in the natural counterparts, portend promising antitumor
properties for the natural products and qualify them as new scaffolds of biological and
medicinal interest. Therefore, the described chemistry highlights the benefits of the
olefin metathesis reaction in the field of the total synthesis of natural products, featuring
convergency and flexibility for structural diversity and has allowed the identification of
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bioactive compounds with interesting antitumor properties. The completion of the
synthesis of the natural products, the design of new analogues based upon compound 58
,
and further biological studies to elucidate the mechanism of its antitumoral and
antiangiogenic activities are currently in progress and they will be reported in due course.
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EXPERIMENTAL SECTION
General Techniques. All reactions were carried out under an argon atmosphere with dry,
freshly distilled solvents under anhydrous conditions, unless using aqueous reagents or
otherwise noted. All solvents used in reactions were dried and distilled using standard
procedures. Tetrahydrofuran (THF) was distilled from sodium benzophenone, and methylene
chloride (CH
2
Cl
2
) from calcium hydride. Yields refer to chromatographically and
1
spectroscopically ( H NMR) homogeneous materials, unless otherwise stated. All solutions
used in workup procedures were saturated unless otherwise noted. All reagents were purchased
at highest commercial quality and used without further purification unless otherwise stated. All
reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm silica gel plates
(60F-254) using UV light (254 nm) as visualizing agent and acidic ceric ammonium molybdate/
phosphomolybdic acid or potassium permanganate solutions and heat as developing agents.
Flash column chromatography (FCC) was performed using silica gel (60 Å, particle size 230-
4
00 mesh) under air pressure. All solvents used for chromatographic purifications were distilled
1
13
prior to use. H and C NMR spectra were recorded on a Bruker DPX-400 MHz instrument
and calibrated using residual undeuterated solvent as an internal reference. Chemical shifts are
reported in ppm with the resonance resulting from incomplete deuteration of the solvent as the
13
13
internal standard ( CDCl
3
: 7.26 ppm, s and 77.0 ppm, t; CD
3
OD: 4.87 ppm, s, 3.31 ppm, quin
1
3
and 49.1 ppm, sep; C
2
D
6
OS: 2.49 ppm, quin and 39.52 ppm, sep). Data are reported as
1
follows: chemical shift /ppm (multiplicity, coupling constants J (Hz) and integration ( H
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only)). The following abbreviations were used to explain the multiplicities: s = singlet; d =
doublet; t = triplet; q = quartet; quin = quintet; b = broad; m = multiplet or combination thereof.
13C signals are singles, unless otherwise stated. High resolution mass spectrometry (HRMS)
was performed on a H-ESI and APCI mass spectrometer in positive mode and using an ion trap
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(Orbitrap) as the mass analyzer type. HRMS signals are reported to 4 decimal places and are
within  5 ppm of theoretical values. Specific optical rotations were recorded on a Perkin-Elmer
241 polarimeter with a sodium halogen lamp (ƛ = 589 nm) and a cell path length of 100 mm (c
given in g/100 mL). Melting points were collected using a Gallenkamp or a Griffin melting
point system using a gradient of 0.5 ºC per min.
Biological Material and Methods. Bovine aortic endothelial cells (BAEC) were isolated by
collagenase digestion, as previously described,30 and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing glucose (1 g/L) supplemented with 10% FBS
(DMEM/10% FBS). All the cancer cell lines used in this study were obtained from the
American Type Culture Collection (ATCC). Human fibrosarcoma HT-1080, hepatocellular
carcinoma HepG2 and glioblastoma U87MG cells were maintained in Eagle's Minimum
Essential Medium (EMEM) supplemented with 10% FBS. Human colon adenocarcinoma HT-
29 cells and human osteosarcoma U2OS cells were maintained in DMEM containing glucose
(4,5 g/L) supplemented with 10% FBS. Human breast cancer carcinoma MDA-MB-231,
chronic myelogenous leukemia KU812F and histiocytic lymphoma U937 cells were maintained
in RPMI1640 medium supplemented with 10% FBS. Acute promyelocytic leukemia HL-60
cells were maintained in RPMI1640 medium supplemented with 20% FBS. All culture medium
contained glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 μg/mL) and amphotericin
(
1.25 μg/mL) and all cell lines were grown at 37 °C and humidified 5% CO atmosphere.
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