Total synthesis and biological evaluation of fluorinated cryptophycins

Article abstract of DOI:10.3762/bjoc.8.231

Cryptophycins are cytotoxic natural products that exhibit considerable activities even against multi-drug-resistant tumor cell lines. As fluorinated pharmaceuticals have become more and more important during the past decades, fluorine-functionalized cryptophycins were synthesized and evaluated in cell-based cytotoxicity assays. The unit A trifluoromethyl-modified cryptophycin proved to be highly active against KB-3-1 cells and exhibited an IC ₅₀ value in the low picomolar range. However, the replacement of the 3-chloro-4-methoxyphenyl-substituent in unit B by a pentafluorophenyl moiety resulted in a significant loss of activity.

Full text of DOI:10.3762/bjoc.8.231

Total synthesis and biological evaluation of
fluorinated cryptophycins
Christine Weiß, Tobias Bogner, Benedikt Sammet and Norbert Sewald*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 2060–2066.
Organic and Bioorganic Chemistry, Department of Chemistry,
Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany
doi:10.3762/bjoc.8.231
Received: 04 September 2012
Accepted: 09 November 2012
Published: 23 November 2012
Email:
Norbert Sewald* - norbert.sewald@uni-bielefeld.de
* Corresponding author
This article is part of the Thematic Series "Antibiotic and cytotoxic
peptides".
Keywords:
antimitotic drug; cytotoxicity; depsipeptide; fluorinated natural product
analogues; structure-activity relationship
Associate Editor: D. Dixon
© 2012 Weiß et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Cryptophycins are cytotoxic natural products that exhibit considerable activities even against multi-drug-resistant tumor cell lines.
As fluorinated pharmaceuticals have become more and more important during the past decades, fluorine-functionalized crypto-
phycins were synthesized and evaluated in cell-based cytotoxicity assays. The unit A trifluoromethyl-modified cryptophycin proved
to be highly active against KB-3-1 cells and exhibited an IC50 value in the low picomolar range. However, the replacement of the
3-chloro-4-methoxyphenyl-substituent in unit B by a pentafluorophenyl moiety resulted in a significant loss of activity.
Introduction
Cryptophycins form a class of cytotoxic sixteen-membered
macrocyclic depsipeptides. Cryptophycin-1 (1) was isolated for
the first time in 1990 from cyanobacteria Nostoc sp.
ATCC 53789 [1] (Figure 1). Moore et al. isolated cryptophycin-
1 from the related Nostoc strain GSV 224, investigated the
stereochemistry, and described the cytotoxicity [2]. At the same
time Kobayashi et al. succeeded in a full structural analysis and
described the first total synthesis of another member of the
cryptophycin family [3,4]. Twenty-eight naturally occurring
cryptophycins have been isolated up to this day [5-7], while
numerous synthetic analogues have been synthesized in the
Figure 1: Structures of cryptophycin-1 (1) and -52 (2).
frame of structure–activity-relationship studies [8,9]. Crypto-
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phycins display remarkable biological activity against multi- Unit A para-alkoxymethyl derivatives of cryptophycin-52 have
drug-resistant (MDR) tumor cells. Such tumor cells express a been synthesized and were shown to retain cytotoxicity even
P-glycoprotein, a drug efflux pump that transports xenobiotics against MDR tumor cell lines [19]. The introduction of a fluo-
out of the cell. A synthetic analogue, cryptohycin-52 (2, rine substituent in the same position also provides a cytotoxic
LY355703), has been investigated in clinical trials. However, analogue, albeit with decreased biological activity by a factor of
this development was discontinued because of neurotoxic side 5 [8].
effects and lacking efficacy in vivo [10,11].
In unit B the chlorine and the methoxy substituents at the
Fluorinated drugs are gaining increasing importance, and D-tyrosine residue were crucial for high antimitotic activity
currently about 20% of all pharmaceuticals on the world market [17,18]. Moore et al. patented the synthesis of fluorinated
contain fluorine substituents [12,13]. Fluorination is supposed analogues of cryptophycin-1 and cryptophycin-52 [20]. In
to enhance bioavailability and receptor selectivity. The van der particular, derivative 3 was shown to retain biological activity
Waals-radius of a fluorine substituent (1.47 Å) lies between the (IC50 = 39 pM) and was active against the tumor cell
value of a hydrogen substituent (1.20 Å) and an oxygen line KB-3-1 [21] (Figure 2). The chlorohydrin derived from 4
substituent (1.52 Å). However, despite this similarity in size, a that also contained a fluorine substituent in the para-position of
fluorine substituent exerts considerable electronic effects due to the unit A phenyl ring was patented as a promising candidate
the high electronegativity. A trifluoromethyl substituted [22].
analogue of epothilone, another important tubulin-binding cyto-
toxic drug, was shown to retain the cytotoxic activity of the In the frame of our on-going SAR studies on cryptophycins
parent compound. At the same time nonspecific side effects due [19,23-30], we envisaged the synthesis of analogues of crypto-
to oxidative degradation were prevented by the introduction of phycin-52 with a para-trifluoromethyl substituent at the unit A
the CF3 group [14,15]. Likewise, partially fluorinated taxoids, aryl ring. In addition, we targeted the replacement of the unit B
analogues of paclitaxel and docetaxel, displayed biological by a D-pentafluorophenylalanine residue.
activity even exceeding that of the parent nonfluorinated com-
Results and Discussion
pounds [16]. The interesting biological profile of fluorinated
Cryptophycin-52 with a para-trifluoromethyl
substituted unit A
cytotoxic agents prompted us to synthesize partially fluorinated
analogues of cryptophycins.
The synthesis of the para-trifluoromethyl substituted unit A
started with a modified Knoevenagel condensation [23,31]. The
required aldehyde 9 was obtained by DIBAL-H reduction of the
corresponding methyl ester 8 and was found to decompose upon
chromatographic purification (Scheme 1). However, it can
usually be employed in the Knoevenagel condensation without
purification. Reaction of 9 with malonic acid in the presence of
piperidine/acetic acid gave the β,γ-unsaturated carboxylic acid
10. The latter compound was transformed into the methyl ester
by treatment with SOCl2 in methanol. The resulting ester 11
could then be directly employed without purification in the
The depsipeptidic character of the cryptophycins suggests four
different fragments to be assembled in the total synthesis,
named unit A–D (Figure 1). Unit A is an α,β-unsaturated
δ-hydroxy acid that usually also contains a benzylic epoxide or
a benzylic double bond. Unit B represents a chlorinated
O-methyl-D-tyrosine derivative, while unit C is a β2-amino
acid, usually β2-homoalanine. Finally, unit D is leucic acid, the
hydroxy analogue of leucine. Numerous synthetic analogues
have been obtained in the frame of structure–activity-relation-
ship studies (SAR-studies), as reviewed in [17,18].
Figure 2: Fluorinated derivatives of cryptophycin-1 and -52 [20-22].
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Beilstein J. Org. Chem. 2012, 8, 2060–2066.
Scheme 1: Access to the trifluoromethyl substituted unit A-building block 16. Reagents and conditions: (a) SOCl2, MeOH, 0 °C → rt, 16 h; (b)
DIBAL-H, CH2Cl2, −78 °C, 3.5 h; (c) malonic acid, piperidine, AcOH, DMSO, 65 °C, 1.5 h; (d) SOCl2, MeOH, 0 °C → rt, 1 h; (e) K2CO3,
K2OsO4∙2H2O, K3[Fe(CN)6], (DHQD)2-PHAL, CH3SO2NH2, t-BuOH/H2O, 0 °C, 42 h; (f) LDA, MeI, THF, −78 °C, 3 d; (g) (CH3)2C(OCH3)2, MeOH,
Amberlyst-15®, rt, 8 d; (h) DIBAL-H, CH2Cl2, −78 °C, 4.5 h; (i) AllylSnBu3, MgBr2∙Et2O, CH2Cl2, −78 °C, 15 h.
asymmetric dihydroxylation with osmium tetroxide and subsequent Yamaguchi esterification of 18 with the unit C–D
(DHQD)2PHAL, in close analogy to a previously published segment 19 and was removed on this stage [33]. Fmoc cleavage
procedure [23]. The initially formed vicinal diol cyclizes under of the seco-depsipeptide 20 liberated the free amino group of
the reaction conditions to give lactone 12 in enantiomerically unit C, which under the reaction conditions displaced the
pure form (chiral HPLC: Chiralpak OD®). Deprotonation of 12 trichloroethylester of unit B resulting in macrocyclization
with 2.5 equiv of LDA, followed by treatment with according to Moher et al. [34]. In the final steps the dioxolane
iodomethane furnished the α-methyl substituted lactone 13. ring of 21 was cleaved with trifluoroacetic acid in the presence
Treatment of this compound with acetone dimethyl acetal in of water. The resulting vicinal diol was not purified, but reacted
methanol in the presence of an acidic ion exchanger resulted in with a large excess of trimethyl orthoformate. The cyclic
acetonide protection of the vicinal diol, accompanied by methyl orthoester resulting from this transformation was directly
ester formation. The methyl ester 14 was subsequently reduced subjected to reaction with acetyl bromide to form a bromohy-
with DIBAL-H to give the aldehyde 15. In order to avoid drin formate. This was then treated with a potassium carbonate/
epimerisation, this aldehyde was not purified, but filtered ethylene glycol/dimethoxyethane-emulsion to bring about
through Celite only and then reacted with allyl-tri-n-butyltin to cleavage of the formyl ester accompanied by epoxide forma-
give the homoallyl alcohol 16. The magnesium bromide diethyl tion as previously described by us [19]. The trifluoromethyl
etherate mediated allylation proceeded under substrate control substituted cryptohycin-52 analogue 22 was obtained in a yield
and with complete diastereoselectivity [23,32].
of 39% over the final four steps. It was purified by column
chromatography, followed by lyophilization.
Cross-metathesis of homoallyl alcohol 16 with the unit B
derived acrylamide 17 provided the α,β-unsaturated δ-hydroxy
Cryptophycin-52 with D-pentafluorophenyl-
carboxamide 18 (Scheme 2). In order to bring about complete alanine as unit B
metathesis of 16, the acrylamide 17 had to be employed in 1.2- The N-acryloyl derivative 26 of D-pentafluorophenylalanine
fold excess, which resulted in a contamination of the cross- was obtained by carbodiimide esterification of commercially
metathesis product 18 with minor amounts of the homo- available Boc-D-pentafluorophenylalanine (24) with
coupling product 23. The latter could not be separated by flash trichloroethanol, followed by cleavage of Boc and reaction with
chromatography on this stage, but did not interfere with the acryloylchloride in the presence of base [19] (Scheme 3).
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Scheme 2: Assembly of units A–D and macrocyclization, followed by diol-epoxide transformation to give the trifluoromethyl substituted analogue 22 of
cryptophycin-52. Reagents and conditions: (a) Grubbs II catalyst, CH2Cl2, reflux, 16 h; (b) 19, DMAP, NEt3, 2,4,6-trichlorobenzoylchloride, THF, 0 °C,
1 h; (c) piperidine, DMF, rt, 16 h; (d) TFA, CH2Cl2, H2O, 0 °C, 3 h; (e) (CH3O)3CH, PPTS, CH2Cl2, rt, 2 h; (f) AcBr, CH2Cl2, rt, 4 h; (g) K2CO3, DME,
ethylene glycol, rt, 3 min.
Scheme 3: Synthesis of the pentafluorophenylalanine building block 26. Reagents and conditions: (a) pyridine, trichloroethanol, DCC, CH2Cl2, 0 °C,
20 h; (b) 1. TFA, rt, 2 h; 2. NEt3, acryloylchloride, CH2Cl2, 0 °C, 7 h.
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The cryptophycin analogue with D-pentafluorophenylalanine as
unit B was synthesized by the same convergent route as
described for derivative 22. Homoallyl alcohol 27 [23] was
reacted with the D-pentafluorophenylalanine derivative 26 in a
cross-metathesis reaction in the presence of Grubbs II catalyst
(Scheme 4). The resulting α,β-unsaturated δ-hydroxy carbox-
amide 28, representing units A and B was then esterified with
19 under Yamaguchi conditions with 2,4,6-trichloroben-
zoylchloride and triethylamine in the presence of catalytic
amounts of DMAP. Macrocyclization was brought about by
Table 1: Cytotoxicity of the fluorinated cryptophycins 22 and 31 in
comparison to cryptophycin-52 (2).
IC50 [pM]
(KB-3-1)
IC50 [nM]
(KB-V1)
FR
2
15.5
66.0
2970
0.26
10.1
98.4
16.7
153
33
22
31
cleavage of the Fmoc protecting group from the unit C amino tumor cell line KB-3-1 and its MDR correlate KB-V1. The IC50
group, which concomitantly displaced the trichloroethylester at values of the fluorinated cryptophycins 22 and 31 were
unit B to result in the macrocyclic product 30 [34]. Cleavage of compared to cryptophycin-52 in Table 1 [17]. While the cyto-
the dioxolane liberated the vicinal diol, which was then toxicity of the unit A-modified analogue 22 against the tumor
subjected to the final diol-epoxide transformation to provide the cell line KB-3-1 was only by about a factor of 4 decreased
cryptophycin-52 analogue 31 in a yield of 14% over the final compared to cryptophycin-52, the pentafluorophenylalanine-
four steps.
containing derivative 31 was much less active. A significant
loss in activity of both analogues against the MDR cell line
The biological activities of the fluorine-functionalized crypto- KB-V1 was observed. The degree of activity against MDR
phycin analogues were determined in a resazurin assay with the tumor cells can be described by the resistance factor FR, which
Scheme 4: Convergent synthesis of the pentafluorinated cryptophycin 31. Reagents and conditions: (a) Grubbs II catalyst, CH2Cl2, reflux, 16 h; (b)
19, DMAP, NEt3, 2,4,6-trichlorobenzoylchloride, THF, 0 °C, 1 h; (c) piperidine, DMF, rt, 16 h; (d) TFA, CH2Cl2, H2O, 0 °C, 3 h; (e) (CH3O)3CH, PPTS,
CH2Cl2, rt, 2 h; (f) AcBr, CH2Cl2, rt, 4 h; (g) K2CO3, DME, ethylene glycol, rt, 3 min.
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The synthesis of selectively fluorinated cryptophycin-52
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obtained. The two analogues were less active, both against the
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Acknowledgements
We thank A. Nieß, L. Ahlers, and C. Michalek for technical
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