Utilization of achiral alkenyl amines for the preparation of high affinity Grb2 SH2 domain-binding macrocycles by ring-closing metathesis

Article abstract of DOI:10.1039/b611887a

A family of previously reported ring-closing metathesis (RCM)-derived macrocycles that exhibit potent Grb2 SH2 domain-binding affinity is characterized by stereoselectively-introduced upper ring junctions that bear bicyclic aryl substituents. However, the synthetic complexity of these macrocycles presents a potential limit to their therapeutic application. Therefore, the current study was undertaken to simplify these macrocycles through the use of achiral 4-pentenylamides as ring-forming components. A series of macrocycles (5a-f) was prepared bearing both open and cyclic constructs at the upper ring junction. The Grb2 SH2 domain-binding affinities of these macrocycles varied, with higher affinities being obtained with cyclo-substituents. The most potent analogue (5d) contained a cyclohexyl group and exhibited Grb2 SH2 domain-binding affinity (K_D = 1.3 nM) that was nearly equal to the parent macrocycle (2), which bore a stereoselectively- introduced naphthylmethyl substituent at the upper ring junction (K_D = 0.9 nM). The results of this study advance design considerations that should facilitate the development of Grb2 SH2 domain-binding antagonists. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b611887a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Utilization of achiral alkenyl amines for the preparation of high affinity Grb2
SH2 domain-binding macrocycles by ring-closing metathesis†
Fa Liu,a Karen M. Worthy,b Lakshman Bindu,b Alessio Giubellino,c Donald P. Bottaro,c Robert J. Fisherb and
Terrence R. Burke, Jr.*a
Received 17th August 2006, Accepted 28th September 2006
First published as an Advance Article on the web 4th December 2006
DOI: 10.1039/b611887a
A family of previously reported ring-closing metathesis (RCM)-derived macrocycles that exhibit potent
Grb2 SH2 domain-binding affinity is characterized by stereoselectively-introduced upper ring junctions
that bear bicyclic aryl substituents. However, the synthetic complexity of these macrocycles presents a
potential limit to their therapeutic application. Therefore, the current study was undertaken to simplify
these macrocycles through the use of achiral 4-pentenylamides as ring-forming components. A series of
macrocycles (5a–f) was prepared bearing both open and cyclic constructs at the upper ring junction.
The Grb2 SH2 domain-binding affinities of these macrocycles varied, with higher affinities being
obtained with cyclo-substituents. The most potent analogue (5d) contained a cyclohexyl group and
exhibited Grb2 SH2 domain-binding affinity (KD = 1.3 nM) that was nearly equal to the parent
macrocycle (2), which bore a stereoselectively-introduced naphthylmethyl substituent at the upper ring
junction (KD = 0.9 nM). The results of this study advance design considerations that should facilitate
the development of Grb2 SH2 domain-binding antagonists.
peptide mimetics exemplified by 1 that are characterized by a C-
Introduction
terminal (3-bicyclicarylpropyl)amide9 and a bend-inducing pTyr +
1 Ac6c (1-aminocyclohexanecarboxylic acid) residue (Fig. 1).10
We had previously prepared macrocycle 2 as a conformationally
constrained variant of 1 using ring-closing metathesis (RCM)
chemistries (Fig. 1).11,12 Macrocycles based on 2 represent a new
family of highly potent Grb2 SH2 domain-binding antagonists
that can provide single digit nanomolar binding constants even
when mono-anionic phosphoryl mimetics are employed.13 In
designing 2 we hypothesized that introduction of the ring-closing
propenyl segment into 1 could enhance binding affinity in part due
to rotational restriction at the upper ring junction. However, when
macrocycle 3 was prepared bearing both R and S stereochemistries
at the upper ring junction, little difference was observed in the
binding affinities (Fig. 1).14 The related RCM-derived macrocycle
4 also showed a surprising lack of dependence of binding affinity
on upper ring junction stereochemistry.15,16 Therefore, a study was
undertaken to prepare a series of macrocycles 5a–5f lacking a
stereogenic center at the upper ring junction and to examine their
Grb2 SH2 domain binding affinities.
Inhibition of protein-tyrosine kinase (PTK)-dependent signaling
has emerged as an important new approach toward anticancer
therapeutics.1,2 Most clinically effective small molecule PTK
inhibitors interact at the kinase catalytic cleft, where they function
as ATP-competitive agents. Although PTK inhibitors generally
exhibit low collateral cytotoxicity, the cardiotoxicity recently
reported following administration of the c-Abl PTK inhibitor,
imatinib mesylate (Gleevec)3 highlights a need for alternate
approaches toward PTK down regulation.
Src homology 2 (SH2) domain binding interactions are key
components of PTK pathways that function downstream of the
kinase step and afford potentially complimentary targets for
blocking PTK signaling.4 Development of SH2 domain binding
inhibitors, including inhibitors of the growth factor receptor
bound protein 2 (Grb2) SH2 domain, has been undertaken by
several groups.5,6 Grb2 is a non-catalytic docking module that
plays critical roles in several cancers, including erbB-2 dependent
breast cancers and c-Met dependent renal carcinomas.7 The design
of Grb2 SH2 domain signaling inhibitors has been assisted by
the preferential binding of native “pTyr-Xxx-Asn-Yyy” ligands in
type-I beta turn conformations.8 A group at Novartis developed
Results and discussion
Synthesis
aLaboratory of Medicinal Chemistry, Bldg. 376 Boyles St., Center for Cancer
Research, NCI-Frederick, National Institutes of Health, Frederick, MD
21702, USA. E-mail: tburke@helix.nih.gov; Fax: 301-846-6033; Tel: 301-
846-5906
A key feature of macrocycles 5a–5f is the facile synthesis of
achiral 4-penten-1-amines (9a–9f) that serve as the upper ring
junction components during RCM ring closure (Scheme 1). With
the exception of 4-penten-1-amine (9a), which was derived from
4-penten-1-ol,17 amines 9b–9f were prepared by the method of
Bender and Widenhoefer, namely by allylation of the nitriles 6b–
6f to yield the corresponding known 1-(2-propenyl)carbonitriles
(8b–8f) followed by reduction.18 For analogue 9d, commercially
available cyclohexanecarbonitrile (6d) was first halogenated to the
bProtein Chemistry Laboratory, Bldg. 469, SAIC-Frederick, Frederick, MD
21702, USA
cUrologic Oncology Branch, National Cancer Institute, National Institutes
of Health, Bethesda, MD 20989, USA
† Electronic supplementary information (ESI) available: Procedures for
the whole cell Grb2 binding studies shown in Fig. 2 as well as synthetic
experimental details for compounds 9c, 9e, 9f, 10b, 10c, 10e, 10f, 13b, 13c,
13d, 13e, 13f, 5b, 5c, 5d, 5e and 5f. See DOI: 10.1039/b611887a
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Scheme 1 Reagents and conditions: (i) CH3SO2Cl, NEt3, CH2Cl2, room
temp, 3 h; then NaN3, DMF–H2O, 50 ◦C, overnight; (ii) LiAlH4, Et2O,
0 ◦C, 1 h then (Boc)2O, Et2O–H2O, room temp, overnight; (iii) CF3CO2H,
Et3SiH, CH2Cl2, room temp, 2 h (52% yield over three steps); (iv)
LiN(i-Pr)2, THF, −78 ◦C, 45 minutes, then allyl bromide, warmed to room
temp overnight; for 8f; NaH, DMF 0◦ to room temp, overnight (65% yield
for 8b; 84% yield for 8c; 91% yield for 8e); (v) PCl5, pyridine, CHCl3, reflux
overnight (95% yield); (vi) allyl bromide, n-BuLi, THF, −78 ◦C, 2 h (76%
yield); (vii) LiAlH4, Et2O for 9b, 9d, 9f; THF for 9c, 9e, 0 ◦C, 1 h (82%
yield for 9b; 92% yield for 9c; 92% yield for 9e; quantitative over two steps
for 9f).
Scheme
2
Reagents and conditions: For 10a–10c, 10e and 10f,
Fig. 1 Structures of Grb2 SH2 domain-binding inhibitors.
Na-Boc-L-Asn(Trt), 4-methylmorpholine, i-BuOC(O)Cl, DMF, 0 ◦C then
room temp, overnight (30% yield for 10a; 82% yield for 10b; 92% yield
for 10c; 92% yield for 10e; quantitative for 10f). For 10d, Na-Boc-L-Asn,
EDCI, HOBt, NEt(i-Pr)2, CH2Cl2, room temp, overnight (11% yield over
two steps from 8d).
known 1-chloro derivative (7d) by the method of Fleming et al.,
then metalated (n-butyl lithium) and reacted with allyl bromide to
yield the intermediate 8d.19
Initial attempts to couple the cyclohexyl-containing amine
9d with Na-Boc-Asn-OH using HOBt–EDCI active ester
methodology20 gave only a low yield of the desired asparagine
amide 10d. A similar effort at coupling the diphenyl-containing
amine 9f resulted in no product. Alternatively, the mixed anhydride
(i-BuOC(O)Cl, 4-methylmorpholine) coupling of amines 9a–9c
and 9e, 9f with Na-Boc-Asn(Trt)-OH gave the desired asparagine
amides 10 in high yields (Scheme 2).
Previous syntheses of macrocycles exemplified by 2 and 3 have
involved the low yield coupling of the protected b-vinyl-containing
pTyr mimetic 12 with the dipeptides 11, obtained by condensing
the appropriate asparagine alkenylamides with N-protected 1-
aminocyclohexanecarboxylic acid (Scheme 3).12–14,21–23 The poor
yields of this key step can be attributed to steric interactions
between the highly hindered 12 and the dipeptide 11.
sterically less crowded monomeric 1-aminocyclohexanecarboxylic
acid allyl ester (16) was undertaken (Scheme 4). Preparation of
16 was achieved in two steps from commercially-available N-Boc-
1-aminocyclohexanecarboxylic acid (14) by initial O-allylation to
15 (50% yield) followed by TFA cleavage of the N-Boc group.
Without purification, crude 16 was directly reacted with 12 using
HOAt–EDCI active ester coupling24 in the presence of NEt(i-Pr)2
to provide desired product 17 in 90% yield. Cleavage of the O-allyl
ester [Pd(PPh3)4, morpholine] gave the dipeptide equivalent 18 in
91% yield suitably protected for further coupling.
Treatment of the N-Boc protected asparagine alkenylamides
10a–10f with TFA gave the corresponding TFA salts (19a–
19f), which were directly subjected to HOAt–EDCI active
ester coupling with pseudodipeptide 18 in the presence of
NEt(i-Pr)2 (Scheme 5). In this fashion the open-chain metathesis
precursors 13a–13f were uniformly obtained in good to high yields.
In order to overcome the low yields encountered in the
condensation of 12 with the dipeptides 11, coupling of 12 with the
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Table 1 Grb2 SH2 domain-binding affinities determined by surface
plasmon resonance
Compound no.
KD/nMa
2
0.9
320
360
24
1.3
39
5a
5b
5c
5d
5e
5f
72
a Steady state values determined on Biacore 2000 and S51 instruments
using amine coupled surfaces as described in references 15 and 34.
Metathesis ring closure to the intermediate protected
macrocycles 21 using Grubbs 2nd generation catalyst
[((PCy3)(Im(Mes)2)Ru CHPh) 20]25 failed when conducted
=
in refluxing dichloromethane (bp 40 ◦C). However, increasing
the reaction temperature through the use of refluxing 1,2-
dichloroethane (bp 83 ◦C) provided the desired protected
ring-closed intermediates 21a–21f, which were passed down a
silica gel column. The brown products were deprotected using
TFA and purified by reverse phase HPLC to yield the final
products 5a–5f as white solids. Yields of HPLC-purified products
were disappointing, ranging from 4% (for 5f) to 39% (for 5a).
Scheme 3 Previous low yield route to metathesis precursors 13.
Grb2 SH2 domain-binding affinities
Grb2 SH2 domain binding affinities of final macrocycles 5a–
5f were determined by surface plasmon resonance (SPR) using
Biacore 2000 and S51 instruments. While SPR-determination of
SH2 domain binding constants has often been indirect through
the measurement of an inhibitor’s ability to compete with sensor-
bound reference pTyr-containing peptide for binding to Grb2 SH2
domain protein in solution,26–28 our current protocol directly mea-
sured the binding of macrocycles 5a–5f to biotinylated Grb2 SH2
domain protein immobilized onto the sensor chip. Immobilization
of protein was achieved either by amine coupling or by streptavidin
capturing of the biotin functionality. Binding affinities determined
from steady state KD values using amine-coupled surfaces (Table 1)
were generally poorer by up to an order of magnitude or more than
Scheme 4 Reagents and conditions: (i) Allyl bromide, Na2CO3, DMF,
room temperature, 2 days (50% yield); (ii) CF3CO2H, Et3Si, CH2Cl2, room
temp, 2 h; (iii) HOAt, EDCI, NEt(i-Pr)2, DMF, room temp, 2 days (90%
yield over two steps); (iv) Pd(PPh3)4, morpholine, THF, room temp, 30 min
(91% yield).
Scheme 5 Reagents and conditions: (i) HOAt, EDCI, NEt(i-Pr)2, DMF, room temp, 2 days (for 13a 91% yield over two steps from 10a; for 13b 90% yield
over two steps from 10b; for 13c quantitative yield over two steps from 10c; for 13d 60% yield over two steps from 10d; for 13e quantitative yield over two
steps from 10e; for 13f 85% yield over two steps from 10f); (ii) C2H4Cl2, reflux, 2 days; (iii) CF3CO2H, Et3Si, CH2Cl2, room temp, 2 h (for 5a 39% yield
over two steps from 13a; for 5b 11% yield over two steps from 13b; for 5c 7% yield over two steps from 13c; for 5d 17% yield over two steps from 13d; for
5e 6% yield over two steps from 13e; for 5f 4% yield over two steps from 13f.
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the corresponding values obtained from biotin capture surfaces
(data not shown). The reasons for these differences are not known.
The binding affinity of the parent macrocycle 2, which bears
a stereoselectively-introduced naphthylmethyl substituent at the
upper ring junction, was found to be KD = 0.9 nM, in close agree-
ment with previous determinations.12 The affinities of macrocycles
5a (KD = 320 nM) and 5b (KD = 360 nM) that contained either
unsubstituted or bis-methyl substituted upper ring junctions were
significantly less than other members of the series. The highest
affinities were obtained with macrocycles having cyclopentyl and
and absence of epidermal growth factor. A431 cells over-express
the epidermal growth factor receptor (EGFR) PTK. Following
incubation with inhibitor the cells were washed, lysed and binding
of Grb2 to EGFR measured as described in the Electronic
Supplementary Information (ESI).† As shown in Fig. 2, at 300 nM
concentration macrocycle 5c was able to achieve a nearly complete
blockade of intracellular binding of Grb2 to EGFR.
cyclohexyl substituents at the upper ring junctions (5c, KD
=
24 nM and 5d, KD = 1.3 nM, respectively). Increasing the
substituent ring size to seven members resulted in a decrease in
affinity (5e, KD = 39 nM). Macrocycle 5f having a bis-phenyl-
substituted ring junction showed a further loss of affinity (KD
=
72 nM), yet retained greater affinity than the unsubstituted and
bis-methyl-containing macrocycles 5a and 5b, respectively.
In open-chain 1 the naphthyl ring system has been hypoth-
esized to interact with a hydrophobic patch on the Grb2 SH2
domain protein formed by the side chains of LysbD6 and Leu
bDꢀ1 and bounded by Phe bE3.9 Structure–activity studies on
the related phosphopeptides “Ac-pTyr-Ile-Asn-NH-R” showed
an approximate 10-fold loss of binding affinity when R = 3-
(1-naphthyl)propyl was replaced by the less hydrophobic 3-
phenylpropyl, isopropyl or isobutyl groups.9 The structural im-
portance of substituents occupying the naphthyl ring position in
1 has also been shown by affinity enhancements achieved through
the introduction of oxygen functionality onto the naphthyl ring29
or by replacement of the naphthyl group with indole ring systems.30
Molecular modeling had previously indicated that the naphthyl
ring system in 2 should interact with the hydrophobic patch
formed by the side chains of LysbD6 and Leu bDꢀ1.21 A recent X-
ray crystal structure of a similar naphthyl-containing macrocycle
bound to Grb2 SH2 domain protein showed that the naphthyl
ring lay primarily over the Leu bDꢀ1 region with the macrocycle
ring-closing propenyl bridge interacting with the methylenes of
the LysbD6 side chain.31 Because macrocycles 5a–5f lack the
naphthylmethyl substituent of parent 2, reduced interactions with
the hydrophobic patch would be expected relative to 2, although
hydrophobic interactions with the LysbD6 side chain would be
retained. Therefore, note was taken of the fact that cyclohexyl-
containing 5d retained nearly all the affinity of the parent 2, which
contained a stereoselectively introduced naphthylmethyl group at
the upper ring junction.
Fig. 2 Concentration-dependent inhibition by 5c of binding of Grb2 to
epidermal growth factor receptor (EGFR) tyrosine kinase in intact A431
cells as described in the Electronic Supplementary Information.† Cells
were treated with the indicated concentrations of 5c, then left untreated
(control) or briefly stimulated with EGF (+EGF). Cells were lysed and the
amount of EGFR bound to Grb2 determined (upper panels) as well as the
amount of total Grb2 (lower panels).
Conclusions
Macrocycles exemplified by 2 exhibit promising Grb2 SH2 domain
inhibitory profiles. However, their synthetic complexity presents
potential limitations to their therapeutic application. The goal of
the current study was to employ achiral 4-pentenylamides as ring-
forming components in the RCM formation of a new family of
macrocycles that do not require stereoselective construction of the
upper ring junction. In the course of this work it was found that
the low synthetic yields associated in the original synthesis with
the key coupling of the pTyr mimetic portion with an Ac6c-Asn-
alkenylamide dipeptide unit could be overcome by preconstruction
of a pseudo-dipeptide formed from the pTyr mimetic and the Ac6c
residue and then coupling this with the Asn-akenylamide residue.
Unfortunately, yields of RCM macrocyclization for this series were
disappointingly low. The Grb2 SH2 domain-binding affinities of
these macrocycles varied, depending on the substituents at the
upper ring-forming junction. Higher affinities were obtained with
cycloalkyl substituents, with the most potent analogue having a
cyclohexyl group. It was significant that binding affinity nearly
equivalent to the parent 2 could be obtained without employing
defined chirality at the upper ring junction or using bicyclic aryl
substituents. The results of this study contribute significantly to a
greater understanding of design considerations that may be useful
for the development of Grb2 SH2 domain-binding antagonists.
The unsubstituted 5a and bis-methyl-substituted 5b exhibited
significantly reduced affinity. This could arise from greater flexi-
bility of their macrocycle-forming pentenyl segments and reduced
hydrophobic interactions with the Leu bDꢀ1 region. The greater
affinities of the cyclopentyl (5c) and cyclohexyl (5d)-containing
macrocycles may be attributed to a combination of reduced
conformational flexibility of their pentenyl amide chains and
greater hydrophobic interactions with the Leu bDꢀ1 region. In
spite of its greater lipophilic character, the cycloheptyl-containing
5e may bind with less affinity due to increased conformational
flexibility.
Experimental
2,2-Dimethyl-4-penten-1-amine (9b)
To a suspension of LiAlH4 (5.60 g, 147 mmol) in ether (100 mL)
at 0 ◦C was added dropwise nitrile 8b (4.0 g, 36.7 mmol) and the
reaction mixture was warmed to room temperature and stirred
overnight. To the mixture was cautiously added H2O (25 mL)
and the mixture was vigorously stirred until white. It was dried
It was also of interest to examine the ability of a representative
macrocycle to inhibit the binding of Grb2 to cognate protein
in whole cells. For this purpose, 5c was incubated at different
concentrations with A431 breast cancer cells in the presence
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1
(Na2SO4) and solvent removed to provide previously reported32
9b as a colorless oil (3.20 g, 77% yield). H NMR (400 MHz,
CDCl3) d 5.80 (m, 1 H), 5.03 (m, 2 H), 2.45 (s, 2 H), 1.97 (m, 2 H),
1.55 (m, 2 H), 0.86 (s, 6 H).
a colorless oil (1.00 g, 50% yield). H NMR (400 MHz, CDCl3)
d 5.92 (m, 1 H), 5.25 (m, 1 H), 5.20 (m, 1 H), 4.72 (br s, 1 H),
4.61 (m, 2 H), 2.00 (m, 2 H), 1.84 (m, 2 H), 1.65–1.53 (m, 3 H),
1.53–1.43 (m, 11 H), 1.30 (m, 1 H). FAB-MS (+VE) m/z: 284.2
(M + H)+. HR-FAB (M + H)+: 284.1873, Calc: 284.1862.
1
Na-Boc-N-(triphenylmethyl)-L-asparagine (4-pentenyl)amide (10a)
Dipeptide allyl ester 17
To a solution of Na-Boc-L-Asn(Trt)-OH (950 mg, 2.00 mmol)
in DMF (10 mL) at 0 ◦C with 4-methylmorpholine (1.00 mL,
9.10 mmol) was added isobutylf◦ormate (0.32 mL, 2.45 mmol)
and the mixture was stirred at 0 C (10 minutes) then amine 9a
(478 mg, 2.40 mmol) was added and the reaction mixture was
warmed to room temperature and stirred overnight. The mixture
was diluted with ethyl acetate (150 mL), washed with H2O (2 ×
50 mL), brine (50 mL), dried (Na2SO4) and solvent removed.
Purification by silica gel column chromatography (hexane and
ethyl acetate) afforded 10a as a white solid (325 mg, 30% yield).
1H NMR (400 MHz, CDCl3) d 7.32–7.15 (m, 15 H), 6.98 (br s, 1
H), 6.70 (br s, 1 H), 6.18 (br s, 1 H), 5.77 (m, 1 H), 5.00 (m, 2 H),
4.42 (m, 1 H), 3.21 (m, 2 H), 3.08 (dd, J = 15.0, 3.4 Hz, 1 H), 2.53
(dd, J = 15.0, 2.2 Hz, 1 H), 2.05 (q, J = 7.2 Hz, 2 H), 1.55 (m, 2 H),
1.43 (s, 9 H). FAB-MS (+VE) m/z 542.4 (M + H)+. HR-FABMS
calcd for C33H40N3O4 (M + H)+: 542.3019. Found: 542.3009.
Treatment of Na-Boc-protected 15 (384 mg, 1.35 mmol) with
CF3CO2H (1.56 mL) and triethylsilane (0.64 mL) in CH2Cl2
(3.00 mL) at room temperature (2 h) and removal of volatiles pro-
vided the free amine as its CF3CO2H salt (16). This was dissolved
in DMF (2.0 mL) and added to a pre-formed active ester solution
obtained by reacting pTyr mimetic 1212 (450 mg, 0.90 mmol) in
DMF (4.0 mL) along with HOAt (183 mg, 1.35 mmol), EDCI
(258 mg, 1.35 mmol) and NEt(i-Pr)2 (0.78 mL, 4.50 mmol) with
stirring at room temperature (15 minutes). The combined reaction
mixture was stirred at room temperature (2 days). The mixture
was diluted with ethyl acetate (150 mL), washed with H2O (2 ×
50 mL) and brine (50 mL), dried (Na2SO4) and solvent evaporated.
Purification by silica gel column chromatography (hexane and
ethyl acetate) provided 17 as a white solid (540 mg, 90% combined
1
yield). H NMR (400 MHz, CDCl3) d 7.22–7.10 (m, 4 H), 5.94
(s, 1 H), 5.83–5.77 (m, 2 H), 5.25–5.00 (m, 4 H), 4.46 (m, 2 H),
3.52 (m, 1 H), 3.00 (m, 1 H), 2.92 (d, J = 21.2 Hz, 2 H), 2.52
(m, 2 H), 1.70–1.20 (m, 36 H), 0.85 (m, 1 H). FAB-MS (+VE)
m/z: 662.4 (M + H)+. HR-FABMS calcd for C36H56NNaO4P (M
+ Na)+: 684.3641. Found: 684.3644.
Na-Boc-N-(triphenylmethyl)-L-asparagine
1-(2-propenyl)-cyclohexanemethanamide (10d)
To a solution of 7d (3.80 g, 25.5 mmol) in dry ether (200 mL) at
0
◦C, was cautiously added LiAlH4 (2.0 g, 52.6 mmol) and the
mixture was warmed to room temperature and stirred overnight.
The reaction mixture was cooled to 0 ◦C and quenched by the
cautious addition of H2O (5.0 mL). The mixture was stirred
vigorously until the color became white, then it was dried (Na2SO4)
and solvent evaporated to yield crude amine 9d as a colorless oil
(3.20 g). An aliquot (0.75 g, 5.0 mmol) was added to a pre-formed
active ester solution formed by reacting at room temperature
for 15 minutes Na-Boc-L-Asn-OH (1.16 g, 5.0 mmol), HOBT
(0.70 g, 5.0 mmol), EDCI (1.00 g, 5.0 mmol) and NEt(i-Pr)2
(1.75 mL, 10 mmol) in dry CH2Cl2 (25 mL). The resulting reaction
mixture was stirred at room temperature overnight. The mixture
was diluted with ethyl acetate (200 mL) and washed with H2O
and brine, dried (Na2SO4) and solvent evaporated. Purification
by silical gel column chromatography (hexane and ethyl acetate)
afforded 10d as a colorless solid (200 mg, 11% yield from 7d). 1H
NMR (400 MHz, CDCl3) d 6.94 (s, 1 H), 6.29 (s, 1 H), 6.20 (d, J =
6.4 Hz,1 H), 5.84 (m, 1 H), 5.64 (s, 1 H), 5.10 (m, 2 H), 4.45 (m, 1
H), 3.17 (m, 2 H), 2.94 (dd, J = 15.2, 3.6 Hz, 1 H), 2.55 (dd, J =
15.4, 6.2 Hz, 1 H), 2.03 (m, 2 H), 1.50–1.20 (m, 19 H). FAB-MS
(+VE) m/z: 368.4 (M + H)+. HR-FABMS calcd for C19H34N3O4
(M + H)+: 368.2549. Found: 368.2550.
Dipeptide acid 18
To a solution of dipeptide allyl ester 17 (540 mg, 0.82 mmol)
in anhydrous THF (30 mL) which had been degassed under
argon for 5 minutes was added Pd(PPh3)4 (93 mg, 0.080 mmol)
and morpholine (0.70 mL, 8.00 mmol). The mixture stirred at
room temperature (30 minutes) then 0.1 M HCl (100 mL) was
added, THF was removed in vacuo and the remaining residue was
extracted with ethyl acetate (3 × 50 mL). The combined ethyl
acetate extracts were washed with brine (50 mL), dried (Na2SO4)
and purified by silica gel column chromatography (CH2Cl2 and
methanol) to afford 18 as a white solid (460 mg in 91% yield). 1H
NMR (400 MHz, CDCl3) d 7.21–7.12 (m, 4 H), 5.91 (m, 1 H),
5.75 (s, 1 H), 5.15 (m, 2 H), 3.49 (m, 1 H), 3.10–2.90 (m, 3 H),
2.72–2.55 (m, 2 H), 1.70 (m, 2 H), 1.60–1.25 (m, 33 H), 1.15 (m, 2
H). FAB-MS (+VE) m/z: 644 (M + Na)+. HR-FABMS calcd for
C33H52NNaO8P (M + Na)+: 644.3328. Found: 644.3359.
Metathesis precursor 13a
Na-Boc-protected asparagine amide 10a (94 mg, 0.174 mmol) was
treated with CF3CO2H (1.60 mL) and triethylsilane (0.30 mL)
in CH2Cl2 (1.0 mL) at room temperature (4 h), then volatiles
were removed in vacuo and the residue was placed in a vacuum
(30 minutes) to yield the CF3CO2H amine salt 19a. This was
dissolved in DMF (2.0 mL) and added to a pre-formed active
ester solution prepared by stirring dipeptide acid 18 (70 mg,
0.116 mmol) in DMF (2.0 mL) with HOAT (19 mg, 0.140 mmol),
EDCI (28 mg, 0.140 mmol) and NEt(i-Pr)2 (91 lL, 0.522 mmol)
at room temperature (15 minutes). The resulting reaction mixture
was stirred at room temperature (2 days) then diluted with ethyl
Na-Boc-a-aminocyclohexanecarboxylic acid 2-propenyl ester (15)
A
mixture of Na-Boc-1-aminocyclohexanecarboxylic acid33
(1.70 g, 7.00 mmol), allyl bromide (0.89 mL, 10.5 mmol) and
Na2CO3 (881 mg, 10.5 mmol) in DMF (20 mL) was stirred at room
temperature (2 days). The reaction mixture was diluted with ethyl
acetate (150 mL), washed with H2O (2 × 50 mL) and brine (50 mL),
dried (Na2SO4) and solvent evaporated. Purification by silica gel
column chromatography (hexane and ethyl acetate) afforded 15 as
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acetate (150 mL), washed with H2O (2 × 50 mL) and brine
(50 mL) and dried (Na2SO4). Purification by silica gel column
chromatography (CH2Cl2 and methanol) provided 13a (85 mg,
91% yield). 1H NMR (400 MHz, CDCl3) d 7.35 (t, J = 6.0 Hz, 1
H), 7.24 (dd, J = 8.4, 2.0 Hz, 2 H), 7.19 (d, J = 8.4 Hz, 2 H), 7.11
(d, J = 8.4 Hz, 1 H), 6.78 (br s, 1 H), 6.28 (s, 1 H), 5.89–5.76 (m, 2
H), 5.56 (br s, 1 H), 5.12–4.97 (m, 4 H), 4.49 (dd, J = 13.4, 7.0 Hz,
1 H), 3.46 (t, J = 9.4 Hz, 1 H), 3.25 (m, 1 H), 3.18 (m, 1 H), 3.07
(m, 1 H), 3.00 (dd, J = 21.4, 3.4 Hz, 2 H), 2.79 (m, 2 H), 2.64
(m, 2 H), 2.14–2.08 (m, 4 H), 1.78–1.57 (m, 5 H), 1.52–1.27 (m,
29 H), 1.13 (m, 2 H), 0.60 (m, 1 H). FAB-MS (+VE) m/z: 803.5
(M + H)+. HR-FABMS calcd for C42H67N4NaO9P (M + Na)+:
825.4543. Found: 825.4568.
2 A. Arora and E. M. Scholar, J. Pharmacol. Exp. Ther., 2005, 315,
971–979.
3 R. Kerkela, L. Grazette, R. Yacobi, C. Iliescu, R. Patten, C. Beahm,
B. Walters, S. Shevtsov, S. Pesant, F. J. Clubb, A. Rosenzweig, R. N.
Salomon, R. A. V. Etten, J. Alroy, J.-B. Durant and T. Force, Nat. Med.,
2006, 12, 908–916.
4 G. Scapin, Drug Discovery Today, 2002, 7, 601–611.
5 C. Garcia-Echeverria, in Cancer Drug Discovery and Development:
Protein Kinases: From Inhibitors to Useful Drugs, ed. D. Fabbro
and F. McCormick, Humana Press, Inc.: Totowa, NJ, 2005, pp.
31–52.
6 T. R. Burke, Jr., Int. J. Pept. Res. Ther., 2006, 12, 33–48.
7 P. G. Dharmawardana, B. Peruzzi, A. Giubellino, T. R. Burke, Jr. and
D. P. Bottaro, Anti-Cancer Drugs, 2005, 17, 13–20.
8 J. Rahuel, B. Gay, D. Erdmann, A. Strauss, C. Garcia-Echeverria, P.
Furet, G. Caravatti, H. Fretz, J. Schoepfer and M. G. Gruetter, Nat.
Struct. Biol., 1996, 3, 586–589.
9 P. Furet, B. Gay, G. Caravatti, C. Garcia-Echeverria, J. Rahuel,
J. Schoepfer and H. Fretz, J. Med. Chem., 1998, 41, 3442–
3449.
Macrocyclic final product 5a
A solution of tripeptide 13a (50 mg, 0.063 mmol) in 1,2-
dichloroethane (20 mL) was degassed under argon (5 minutes) then
Grubbs 2nd generation catalyst [((PCy3)(Im(Mes)2)Ru CHPh)
10 C. Garcia-Echeverria, B. Gay, J. Rahuel and P. Furet, Bioorg. Med.
Chem. Lett., 1999, 9, 2915–2920.
11 Y. Gao, C.-Q. Wei and T. R. Burke, Jr., Org. Lett., 2001, 3, 1617–
1620.
=
20]24 (26 mg, 0.032 mmol) was added and the mixture was
refluxed (2 days). The mixture was concentrated and purified
by silica gel column chromatography (CH2Cl2 and methanol)
to provide the brown crude product 21a. This was treated with
a mixture of CF3CO2H (9.0 mL), triethylsilane (0.50 mL) and
H2O (0.50 mL) at room temperature (2 h). The solvent was
removed and the residue was purified by reverse phase preparative
HPLC using a Phenomenex C18 column (21 mm diameter ×
250 mm, cat. no: 00G-4436-P0) using a linear gradient from
0% aqueous acetonitrile (0.1% CF3CO2H) to 100% acetonitrile
(0.1% CF3CO2H) over 35 minutes at a flow rate of 10.0 mL min−1
(detection at 225 nm). Lyophilization provided the macrocyclic
final product 5a as a white solid (15 mg, 39% yield from 13a). 1H
NMR (400 MHz, DMSO-d6) d 8.50 (s, 1 H), 8.32 (d, J = 8.0 Hz,
1 H), 7.57 (br s, 1 H), 7.31 (d, J = 8.4 Hz, 2 H), 7.20–7.13 (m, 4
H), 5.78 (dd, J = 15.0, 10.0 Hz, 1 H), 5.56 (m, 1 H), 4.26 (m, 1
H), 4.09 (d, J = 8.4 Hz, 1 H), 3.55 (m, 1 H), 3.28 (d, J = 11.6 Hz,
1 H), 2.93 (d, J = 21.2 Hz, 2 H), 2.83 (dd, J = 15.8, 5.0 Hz, 1
H), 2.76 (m, 1 H), 2.53 (m, 1 H), 2.33 (dd, J = 15.2, 4.8 Hz, 1 H),
2.20–1.95 (m, 4 H), 1.90–1.70 (m, 3 H), 1.60–1.40 (m, 7 H), 1.20
(m, 1 H). FAB-MS (−VE) m/z: 605.2 (M − H)−. HR-FABMS
calcd for C28H40N4O9P (M + H)+: 607.2533. Found: 607.2558.
12 C.-Q. Wei, Y. Gao, K. Lee, R. Guo, B. Li, M. Zhang, D. Yang and T. R.
Burke, Jr., J. Med. Chem., 2003, 46, 244–254.
13 S.-U. Kang, Z.-D. Shi, K. M. Worthy, L. K. Bindu, P. G. Dharmawar-
dana, S. J. Choyke, D. P. Bottaro, R. J. Fisher and T. R. Burke, Jr.,
J. Med. Chem., 2005, 48, 3945–3948.
14 Z.-D. Shi, R. G. Karki, K. M. Worthy, L. K. Bindu, R. J. Fisher and
T. R. Burke, Jr., Chem. Biodiversity, 2005, 2, 447–456.
15 S. Oishi, Z.-D. Shi, K. M. Worthy, L. K. Bindu, R. J. Fisher and T. R.
Burke, Jr., ChemBioChem, 2005, 6, 668–674.
16 S. Oishi, R. G. Karki, Z.-D. Shi, K. M. Worthy, L. Bindu, O. Chertov,
D. Esposito, P. Frank, W. K. Gillette, M. A. Maderia, J. Hartley, M. C.
Nicklaus, J. J. Barchi, Jr., R. J. Fisher and T. R. Burke, Jr., Bioorg. Med.
Chem., 2005, 13, 2431–2438.
17 X. Hu, K. T. Nguyen, V. C. Jiang, D. Lofland, H. E. Moser and D. Pei,
J. Med. Chem., 2004, 47, 4941–4949.
18 C. F. Bender and R. A. Widenhoefer, J. Am. Chem. Soc., 2005, 127,
1070–1071.
19 F. Fleming Fraser, Z. Zhang and P. Knochel, Org. Lett., 2004, 6, 501–
503.
20 Z.-D. Shi, K. Lee, C.-Q. Wei, L. R. Roberts, K. M. Worthy, R. J. Fisher
and T. R. Burke, Jr., J. Med. Chem., 2004, 47, 788–791.
21 W. Konig and R. Geiger, Chem. Ber., 1970, 103, 788–798.
22 Z.-D. Shi, C.-Q. Wei, K. Lee, H. Liu, M. Zhang, T. Araki, L. R. Roberts,
K. M. Worthy, R. J. Fisher, B. G. Neel, J. A. Kelley, D. Yang and T. R.
Burke, Jr., J. Med. Chem., 2004, 47, 2166–2169.
23 Z.-D. Shi, H. Liu, M. Zhang, K. M. Worthy, L. Bindu, D. Yang,
R. J. Fisher and J. T. R. Burke, Bioorg. Med. Chem., 2005, 15, 4200–
4208.
24 L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397–4398.
25 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Org. Lett., 1999, 1,
953–956.
Acknowledgements
26 M. M. Morelock, R. H. Ingraham, R. Betageri and S. Jakes, J. Med.
The authors wish to thank Drs. James A. Kelley and Christopher
Lai of the Laboratory of Medicinal Chemistry, NCI, for mass
spectral data. This research was supported in part by the Intramu-
ral Research Program of the NIH, Center for Cancer Research,
NCI-Frederick. This project has been funded in whole or in part
with federal funds from the National Cancer Institute, National
Institutes of Health, under contract N01-CO-12400. The content
of this publication does not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the US Government.
Chem., 1995, 38, 1309–1318.
27 Z.-J. Yao, C. R. King, T. Cao, J. Kelley, G. W. A. Milne, J. H. Voigt and
T. R. Burke, Jr., J. Med. Chem., 1999, 42, 25–35.
28 Y.-L. Song, J. Tan, X.-M. Luo and Y.-Q. Long, Org. Biomol. Chem.,
2006, 4, 659–666.
29 J. Schoepfer, B. Gay, N. End, E. Muller, G. Scheffel, G. Caravatti and
P. Furet, Bioorg. Med. Chem. Lett., 2001, 11, 1201–1203.
30 J. Schoepfer, H. Fretz, B. Gay, P. Furet, C. GarciaEcheverria, N.
End and G. Caravatti, Bioorg. Med. Chem. Lett., 1999, 9, 221–
226.
31 J. Phan, Z.-D. Shi, T. R. Burke, Jr. and D. S. Waugh, J. Mol. Biol., 2005,
353, 104–115.
32 R. D’Arcy, C. A. Grob, T. Kaffenberger and V. Krasnobajew, Helv.
Chim. Acta, 1965, 49, 185–203.
33 Available from Chem-Impex International, Inc.
34 S. Oishi, R. G Karki, Z.-D. Shi, K. M. Worthy, L. Bindu, O. Chertov,
D. Esposito, P. Frank, W. K. Gillette, M. Maderia, J. Hartley, M. C.
Nicklaus, J. J. Barchi, Jr., J. Fisher Robert and T. R. Burke, Jr., Bioorg.
Med. Chem., 2005, 13, 2431–2438.
References
1 C. Garcia-Echeverria and D. Fabbro, Mini-Rev. Med. Chem., 2004, 4,
273–283.
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The Royal Society of Chemistry 2007
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