Synthesis, kinase activity and molecular modeling of a resorcylic acid lactone incorporating an amide and a trans-enone in the macrocycle

Article abstract of DOI:10.1016/j.tet.2012.04.082

Details for the synthesis of a resorcylic acid lactone (RAL) incorporating a trans-enone and an amide in the macrocyclic ring are provided herein. The sequence included the assembly of three fragments by esterification, olefination, and lactamization. The RAL with the lactam was less potent as an inhibitor of kinases than other RALs investigated. The biological results were rationalized by docking and molecular dynamics simulations of the lactam bound to human ERK2 and comparison with hypothemycin.

Full text of DOI:10.1016/j.tet.2012.04.082

Tetrahedron 68 (2012) 5533e5540
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Synthesis, kinase activity and molecular modeling of a resorcylic acid lactone
incorporating an amide and a trans-enone in the macrocycle
,
,
Carmela Napolitano a, Viraja R. Palwai b, Leif A. Eriksson a c, Paul V. Murphy a
*
a School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland
b
€
€
School of Science and Technology, Orebro University, 70182 Orebro, Sweden
c
€
Department of Chemistry & Molecular Biology, University of Gothenburg, 41296 Goteborg, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Details for the synthesis of a resorcylic acid lactone (RAL) incorporating a trans-enone and an amide in
the macrocyclic ring are provided herein. The sequence included the assembly of three fragments by
esterification, olefination, and lactamization. The RAL with the lactam was less potent as an inhibitor of
kinases than other RALs investigated. The biological results were rationalized by docking and molecular
dynamics simulations of the lactam bound to human ERK2 and comparison with hypothemycin.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 29 December 2011
Received in revised form 15 April 2012
Accepted 23 April 2012
Available online 28 April 2012
Keywords:
Resorcylic acid lactones
Kinase inhibitors
Olefination
Lactamization
1. Introduction
aliphatic and the aromatic portion of these natural products.7 Re-
cent research has demonstrated the importance of the hydroxyl
Kinase inhibition represents one of the most powerful ap-
proaches for the treatment of diseases related to intra-cellular
signaling and cell cycle regulation.1 Progress has been made and
nine small-molecule inhibitors have been approved by the FDA
over the last decade.1b,2 However, the field still faces challenges.
Kinase inhibitors often show off-target activity as well as activity
against the primary intended target.3 Knowledge of the kinase
selectivity profile of small-molecule inhibitors and understanding
the reasons for activity differences can facilitate the design of more
optimally targeted inhibitors.
The resorcylic acid lactone (RAL) based polyketides hypoth-
emycin,4 LL-Z1640-2,5 and L-783,2776 (Fig. 1) are a structurally
unique group of kinase inhibitors, with a built-in selectivity filter.
The selective and powerful inhibition involves the Michael addition
reaction of a protein thiol on to a cis-enone moiety of the macro-
lactone.4 The covalent inactivation of the kinase is largely confined
to w10% of the kinome and specifically to kinases which bear
a conserved cysteine residue. This corresponds to Cys166 in ERK24
and similarly conserved cysteine residues in other kinases.
Structureeactivity relationship (SAR) studies based on hypoth-
emycin and LL-Z1640-2 have included modifications of both the
group at the C-40 position in order for there to be potent kinase
inhibitory activity.7a There has also been the discovery of meta-
bolically stabilized analogues with both in vitro and in vivo acti-
vities.7c,8 As part of investigation of structureeactivity relationships
for the RALs,9,10 we were interested in synthesis of the lactam 1,
where the hydroxyethylene group, which includes the 40-hydroxy
group, is replaced with an amide (Fig. 1). Although 1 contains
a trans-enone, we believed based on precedent10 that it could
participate in a Michael addition reaction with conserved cysteines
of certain kinases. The presence of the amide in the benzomacro-
lactone scaffold would possibly maintain the potential for hydrogen
* Corresponding author. Tel.: þ353 91 492465; e-mail address: paul.v.murphy@
nuigalway.ie (P.V. Murphy).
Fig. 1. Selected examples of natural RALs and 1.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.082

5534
C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
bonding with the backbone of the kinase and potentially mimic
a hydrogen bonding interaction involving the 40-OH of natural
RALs. A synthetic route to 1 is described herein, which enabled the
evaluation of its affinity as an inhibitor of kinases to be compared
with natural RALs.
with boronate 11 catalyzed by Pd(PPh3)2Cl2 efficiently proceeded to
give the ester 4 (77%). Besides 4, a small amount (8%) of the allylic
alcohol derived from MPM protecting group removal was isolated
during this reaction.
Hydrolysis of the ester 4 was followed by the Mitsunobu re-
action of the resulting acid with alcohol 5 to give the MPM-
diether 12 (Scheme 3). A previous process for the preparation of
5 had a relatively modest 68% yield.9 Hence a two-step conversion
of 16 into alcohol 5 was evaluated. Conversion of the alcohol
16 into its MPM ether (10, CSA) followed by removal of the
2. Synthesis
The synthetic route is summarized in Scheme 1 from the alde-
hyde 2, which gives 1 after intermolecular olefination with the
phosphonate 3 and subsequent lactam formation. The functional-
TBS group using TBAF afforded
5 in 85% overall yield.
4-Methoxyphenylmethyl protecting group removal was next
attempted by treating 12 with DDQ (2.5 equiv) in CH2Cl2/buffer of
pH 7.6 (9:1). These conditions led to the formation of the aldehyde
13 in 50% overall yield starting from 4. Oxidation of aldehyde 13
under Pinnick15 conditions was next carried out and followed by
chemoselective protection (TBSCl, Et3N) of the resulting acid to
smoothly afford 14 (84% over two steps). Subsequent treatment of
14 with the DesseMartin periodinane16 in anhydrous CH2Cl2
yielded aldehyde 15.
ized ester
2 was obtained from 4 and 5 via Mitsunobu
esterification.11
Scheme 1. Retrosynthetic analysis.
The synthesis of the aromatic fragment 4 is summarized in
Scheme 2. Phenol 6 was prepared in two steps starting from 2,4,6-
trihydroxybenzoic acid according to Sugawara and co-workers.12
Next the conversion of 6 into the MOM ether followed by trans-
esterification (LiOH, MeOH/H2O) and then reaction with triflic an-
hydride in the presence of pyridine provided 7 (81% over three
steps). The SuzukieMiyaura coupling of the triflate 7 with boronate
11 was then considered. Propargyl alcohol 8 was converted into its
MPM ether in excellent yield (>97%) using the Dudley reagent [2-
(4-methoxybenzyloxy)-4-methylquinoline, 10]13 under the
Paquette conditions (CSA, CH2Cl2, room temperature).14 Sub-
sequent hydroboration of
9 with 4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (pinacol borane) in the presence of a catalytic
amount of dicyclohexylborane at 35 ꢀC afforded 11 in satisfactory
yield (67%). Subsequently the SuzukieMiyaura-type coupling of 7
Scheme 3. Synthesis of aldehyde 15.
With 15 in hand the formation of the macrocycle of 1 could
conceptually be achieved in either of two ways (Scheme 4). The first
(path A) involves amide formation followed by olefination; the
second (path B) involves olefination followed by lactamization.
Phosphonates 19 and 3 were both prepared with a view to estab-
lishing their usefulness for the formation of the macrocycle of 1. The
reaction of the enolate obtained from reaction of bis(2,2,2-
trifluoroethyl)-methylphosphonate with LHMDS at ꢁ98 ꢀC with
esters 1717 and 18 in presence of TMEDA afforded 19 and 3, re-
spectively, in moderate yields (Scheme 4).
Treatment of the aldehyde 15 with the enolate of 19 (Scheme
5) afforded 21 as the E-isomer in modest yield (37%). However,
the enone 21 could not be converted into 1. Staudinger reduction
of 21 using polymer-supported triphenylphoshine led to degra-
dation of the starting material.18 Conditions recently reported by
Vilarrasa and co-workers, using PySSPy as a promoter for direct
amide bond formation from carboxylic acids and azides, failed
and did not lead to a macrocycle or 22.19 The same reaction
conditions were investigated for intermolecular amide bond
formation from 19 and 20 to give 23 but this approach was also
unsuccessful (Scheme 5).
Scheme 2. Synthesis of the aromatic fragment 4.

C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
5535
Scheme 6. Synthesis of 1.
which consequently have potential for covalent conjugation with
RALs and the results are summarized in Table 1.21 The IC50 values
are compared with those for 25 and LL-Z1640-2, which showed
inhibitory activity to these kinases. The lactam 1 inhibited kinases
in the target subgroup and had IC50 values in the micromolar range.
However 1 was generally significantly less potent than 25, which
has an ethylene group instead of the amide but also contains the
trans-enone;10 the introduction of the amide led to a loss of potency
and also to a loss of selectivity within this subgroup of kinases. For
Scheme 4. Possible approaches to 1. Preparation of 3 and 19.
example, 25 is 37 fold more active toward PDGFRa than VEGFR2,
whereas 1 is w1.6 fold less active toward PDGFR than VEGFR2. The
a
RALs 1 and 25 are both substantially less potent than LL-Z1640-2 in
the enzymatic assays.
Table 1
Inhibition of kinases by 1 and 25a
Compd
PDGFR
a
VEGFR2
VEGFR3
MEK1
FLT3
c-kit
Scheme 5. Attempts to prepare 22 and 23.
1
4.4
2.7
5.9
7.2
1.8
8.0
25
0.027
1.0
1.4
22
0.002
0.33
0.0049
1.3
0.0096
As the use of the azide 19 was unproductive, we turned our
attention to the Boc protected glycine derivative 3. Amide bond
formation (path A, Scheme 4) was first investigated. Reaction of the
acyl chloride of 20 (prepared by treating 20 with oxalyl chloride in
DMF/CH2Cl2) with in situ generated amine prepared from 3 by its
reaction with NaI and DIPEA in MeOH/CH3CN provided almost
exclusively unreacted 20 together with decomposition products. A
number of Boc-removal conditions (TFA/CH2Cl2, TBAF/THF, BBr3/
CH2Cl2) were screened with a view to isolate the amine from 3 but
all proved unsuccessful or low yielding, leading to partial hydrolysis
of the phosphonoester group and/or complex mixtures of products.
The difficult removal of the Boc group from 3 and the very poor
accessibility to the desired intermediate led us to exclude amide
formation followed by olefination strategy to generate the macro-
cycle. Investigation of the intermolecular olefination reaction of the
aldehyde 15 with phosphonate 3 and subsequent macrocyclisation
was ultimately successful (Scheme 6). Reacting the anion of the
StilleGennari phosponate 3 (K2CO3, THF, ꢁ10 ꢀC), with aldehyde 15,
led to the isolation of the E-enone 24 (50%); the Z-isomer was not
observed in this reaction as might have been expected.20 Partial
removal of the MOM group from 24 was observed after chro-
matographic purification. The free phenol was usually present at
a level w10% with 24 after this reaction. The complete removal of
the MOM and also the Boc groups from 24 was achieved using TFA.
This was followed by lactamization (EDC, HOBT) to finally give the
desired lactam 1 (30% from 24).
LL-Z1640-2
2.4ꢂ10ꢁ4
4.9ꢂ10ꢁ4
5ꢂ10ꢁ4
a
IC50 values (
m
M) from two independent experiments. No significant inhibition
M for 1 for the following kinases: ERK2,
was observed at concentrations <100
ERK1, GSK3 , GSK3 , PDGFR , VEGFR1.
m
a
b
b
4. Molecular modeling
A molecular modeling study was carried out to investigate the
binding of 1 to human ERK2 in order to try to obtain some insights
into why it may not be as active as LL-Z1640-2 or its close structural
analogue hypothemycin. The X-ray crystal structure of rat ERK2
bound to hypothemycin (PDB entry 3C9W)22 formed a guiding
point for the generation of a model of 1 (Fig. 1) bound with human
ERK2. For docking (using MOE), a homology model of human ERK2
was generated and used throughout. Both the human and rat ERK2
enzyme structures show a high sequence similarity and the active
site residues are highly conserved. The homology model was based
on the X-ray structure of human ERK2 (PDB entry 2E1); the ho-
mology model was generated as the X-ray structure had a missing
loop. Details for generation of this model are given in the
Experimental section. Docking was then carried out and followed
by molecular dynamics (MD) simulations using YASARA.
Compound 1 and hypothemycin were docked into the ATP
binding site of the model of human ERK2 enzyme. The resulting
docked complex showed that 1 had a completely different binding
mode to ERK2 when compared to that of hypothemycin which
orients in the pocket close to Met108 and with the double bond
positions in close proximity to Cys166, thus enabling the formation
of the covalent bond between ligand and protein. The energy
minimized structure of 1 bound to the enzyme (Fig. 2) showed one
3. Biological evaluation
With 1 in hand, it was screened against a panel of 12 kinases, all
of which contained the important conserved cysteine residue and

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C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
6. Experimental section
6.1. General experimental conditions
NMR spectra were recorded on 400 and 500 MHz spectrome-
ters. Chemical shifts are reported relative to internal Me4Si in CDCl3
13
(d
0.0) for 1H and CDCl3
(
d
77.0) for C at 25 ꢀC, unless otherwise
stated. 13C signals were assigned with the aid of DEPT-135, HSQC,
and HMBC. 1H signals were assigned with the aid of COSY. Coupling
constants are reported in hertz. High-resolution mass spectra were
measured using an ESI time-of-flight mass spectrometer and were
measured in positive and/or negative mode as indicated. IR spectra
were recorded on a PerkineElmer spectrum 1000 FT-IR spec-
trometer. Optical rotations were measured with a Schmidt &
Haensch UniPol L1000 polarimeter. TLC were performed on alu-
minum sheets precoated with Silica Gel 60 (HF254, E. Merck) and
spots visualized by UV and charring with vanillin, molybdate, and/
or ninhydrin solutions. Flash column chromatography was carried
out using Silica Gel 60 (0.040e0.630 mm, E. Merck) and using
a stepwise solvent polarity gradient correlated with TLC mobility.
All moisture-sensitive reactions were performed under a nitrogen
atmosphere unless otherwise specified. Anhydrous DMF and pyri-
dine were used as purchased from SigmaeAldrich. Dichloro-
methane and tetrahydrofuran were used as obtained from a Pure
SolvÔ solvent purification system. Petroleum ether is the fraction
with bp 40e60 ꢀC. Kinase assays were conducted as previously
described.24
Fig. 2. Minimized structure of 1 docked into homology model of human ERK2. Hy-
drogen bonds are indicated with black dotted lines.
6.1.1. 4-Methoxy-2-methoxymethoxy-6-trifluoromethanesulfonyloxyb-
enzoic acid methyl ester 7. To a solution of phenol 6 (3.12 g,
13.8 mmol) and N,N-diisopropylethylamine (4.82 mL, 27.7 mmol)
in CH2Cl2 (20 mL) was added MOMCl (1.26 mL, 16.6 mmol). The
resulting mixture was stirred for 15 h at room temperature, then
diluted with CH2Cl2, washed with 1 M aq HCl solution, dried
(Na2SO4), and filtered. The volatile components were removed
under diminished pressure. Chromatography of the residue (pe-
troleum ether/EtOAc, 80:20e50:50) gave the MOM ether in-
termediate as a pale yellow oil (3.72 g, >95%); 1H NMR (500 MHz,
hydrogen bond between Lys54 and the phenol. Apart from the
hydrogen bond with Lys54, no other hydrogen bonding was ob-
served between 1 and ERK2. The methoxy and carbonyl groups
formed hydrogen bonds with water molecules. The minimized
complex of ERK2 and 1 was subjected to molecular dynamics (MD)
for 6 ns. The final structure after MD did not show any additional
hydrogen bonding interactions with the residues in the active site.
The phenol, two carbonyl groups and the eNH group formed hy-
drogen bonds with water molecules. Overall, the major part of 1 is
solvent exposed, which results in a much lower binding energy
(52 kcal molꢁ1) compared to hypothemycin (72 kcal molꢁ1) for the
non-covalent complexes. The distance between the reactive
carbon of the enone and the sulfur atom of the cysteine residue
CDCl3)
d
1.70 (s, 6H), 3.53 (s, 3H), 3.82 (s, 3H), 5.30 (s, 2H), 6.13 (d, J
166.2
1.5 Hz, 1H), 6.43 (d, J 1.5 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d
(C), 160.5 (C), 159.1 (C), 158.0 (C), 105.0 (C), 97.5 (C), 97.2 (CH), 95.0
(two signals, CH2 and CH), 56.6 (CH3), 55.7 (CH3), 25.6 (2ꢂCH3);
HRMS calcd for C13H16NaO6 [MþNa]þ 291.0845, found 291.0861.
Lithium hydroxide (0.670 g, 27.8 mmol) was added to a stirred
suspension of this intermediate (3.72 g,13.9 mmol) in a 2:1 mixture
MeOH/H2O (36 mL). The reaction mixture was stirred for 90 min at
room temperature, then acidified with 1 M aq HCl solution and
extracted three times with EtOAc. The combined organic portions
were dried (Na2SO4), filtered, and volatiles removed under di-
minished pressure. The methyl ester intermediate was obtained as
a white solid and was used in the next step without further puri-
ꢀ
(Cys166) for the final hypothemycineERK2 complex was 4.6 A.
The corresponding distance in the 1eERK2 complex was approxi-
ꢀ
mately 6 A. This showed that hypothemycin is closer to the cysteine
residue, which can thus aid in the Michael addition reaction
leading to covalent bond formation to the cysteine in the active
site and higher affinity for ERK2. These modeling generated
observations provide an explanation for the low affinity observed
for 1 for ERK2.
fication; 1H NMR (500 MHz, CDCl3)
(s, 3H), 5.18 (s, 2H), 6.17 (s, 2H), 11.87 (s, 1H); 13C NMR (125 MHz,
CDCl3) 171.4 (C), 165.5 (C), 165.1 (C), 159.5 (C), 97.5 (C), 95.1, 95.0
d 3.51 (s, 3H), 3.80 (s, 3H), 3.92
5. Summary and conclusions
d
The synthesis of a new resorcylic acid lactone lactam has been
achieved providing a basis for the generation of new analogues of
RALs for biological evaluation. Although 1 was not a very efficient
inhibitor of kinases the study contributes to the structureeactivity
relationships for kinases. The modeling study provides a rationale
for the lower affinity of 1. Modeling of the cis-enone isomer of 1 to
ERK2 (not shown herein) does indicate it should be a better in-
hibitor of ERK2. The synthesis of this isomer is currently being in-
vestigated. Such benzomacrolactone scaffolds are also of interest
more widely for generation of compounds for screening and the
synthetic work described herein provides a basis for further re-
search in this regard.23
(CH2 and CH), 94.8 (CH), 56.4 (CH3), 55.5 (CH3), 52.2 (CH3); HRMS
calcd for C11H15O6 [MþH]þ 243.0869, found 243.0862. Triflic an-
hydride (3.01 mL, 17.9 mmol) was added dropwise to a stirred so-
lution of this methyl ester in dry pyridine (20 mL). The resulting
mixture was stirred at room temperature for 3 h and EtOAc was
then added. The organic layer was washed twice with 1 M aq HCl
solution, then dried (Na2SO4), filtered, and the solvent was re-
moved under diminished pressure. Chromatography of the residue
(petroleum ether/EtOAc, 85:15e60:40) afforded 7 as an off-white
solid (4.20 g, 81% over two steps); 1H NMR (500 MHz, CDCl3)
d
3.50 (s, 3H), 3.83 (s, 3H), 3.91 (s, 3H), 5.21 (s, 2H), 6.49 (br s, 1H),
6.76 (br s, 1H); 13C NMR (125 MHz, CDCl3)
d
163.3 (C), 162.3 (C),

C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
5537
157.4 (C), 147.9 (C), 118.5 (q, J 318.5, CF3), 110.8 (C), 101.5 (CH), 101.0
(CH), 95.2 (CH2), 56.5 (CH3), 55.9 (CH3), 52.5 (CH3); HRMS calcd for
C12H13F3NaO8S [MþNa]þ 397.0181, found 397.0179.
(CH), 55.3 (CH3), 39.6 (CH2), 25.9 (3ꢂCH3), 24.1 (CH3), 18.1 (C), ꢁ4.4
(CH3), ꢁ4.8 (CH3); HRMS calcd for C18H32NaO3Si [MþNa]þ
347.2018, found 347.2023. TBAF (6.40 mL of a 1 M solution in THF,
6.40 mmol) was added to a stirred solution of TBS-ether in dry THF
(15 mL). The resulting mixture was stirred at room temperature for
24 h, then EtOAc was added. The organic layer was washed with
H2O, dried (Na2SO4), filtered, and the solvent evaporated under
reduced pressure. Chromatography of the residue (petroleum
ether/acetone, 90:10e85:15) gave 5 as a pale yellow oil (522 mg,
92%). The analytical data obtained for 5 were consistent with those
reported in literature.25
6.1.2. 4-Methoxy-2-[3-(4-methoxybenzyloxy)-propenyl]-6-
methoxymethoxy-benzoic acid methyl ester 4. A mixture of propargyl
alcohol (1.16 mL, 20.0 mmol) and 2-(4-methoxybenzyloxy)-4-
methylquinoline 10 (11.1 g, 39.8 mmol) was placed under an atmo-
sphere of argon, dissolved in CH2Cl2 (40 mL), and treated with CSA
(462 mg, 1.99 mmol) to afford a clear, bright yellow solution. The
mixture was stirred for 45 h at room temperature, then volatiles
were removed under diminished pressure. Chromatography of the
residue (petroleum ether/EtOAc, 95:5) afforded 9 as a colorless oil
6.1.4. (2S)-4-Hydroxybutan-2-yl 4-methoxy-2-(methoxymethoxy)-6-
[(1E)-3-oxoprop-1-en-1-yl]benzoate 13. Aq NaOH (2 M, 15.0 mL,
(3.43 g, 98%); 1H NMR (500 MHz, CDCl3)
(s, 3H), 4.14 (d, J 2.4 Hz, 2H), 4.54 (s, 2H), 6.88 (d, J 8.6 Hz, 2H), 7.28 (d,
J 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d
2.45 (t, J 2.4 Hz, 1H), 3.80
30.0 mmol) was added to
a stirred solution of 4 (977 mg,
d
159.4 (C), 129.8 (2ꢂCH),
2.43 mmol) in MeOH (15 mL). The resulting mixture was stirred
whilst heating at reflux until the starting material was consumed.
The volatile components were evaporated under reduced pressure;
the aqueous layer was acidified (final pH 5) with 1 M aq HCl solu-
tion, and extracted three times with CHCl3. The combined organic
portions were dried (Na2SO4), filtered, and the solvent was re-
moved under diminished pressure to give a yellow wax containing
the acid intermediate. The crude residue was used in the next step
129.3 (C), 113.8 (2ꢂCH), 79.8 (C), 74.5 (CH), 71.1 (CH2), 56.7 (CH2),
55.3 (CH3). Next the pinacol borane 11 was prepared. Dicyclohex-
ylborane (1 M) was prepared by the addition of cyclohexene
(0.200 mL, 0.002 mmol) to BH3$THF (1 M solution, 1.00 mL,
1.00 mmol) at 0 ꢀC and stirring for 45 min. Dicyclohexylborane
(700 mL of a 1 M solution, 0.700 mmol) was then added in three
portions to a mixture of 9 (1.00 g, 5.68 mmol) and pinacolborane
(1.40 mL, 9.65 mmol). The reaction mixture was stirred for 24 h at
35 ꢀC, before cooling to room temperature and bubbling air through
the reaction for 2 h. Chromatography of the residue (petroleum
ether/EtOAc, 90:10) gave 11 as a pale yellow oil (1.16 g, 67%); IR (neat,
cmꢁ1): 3054, 2981, 2935, 2839, 1644, 1613, 1513, 1359, 1265, 1144,
without further purification; 1H NMR (500 MHz, CDCl3)
d 3.51 (s,
3H), 3.79 (s, 3H), 3.84 (s, 3H), 4.17 (br d, J 5.9 Hz, 2H), 4.51 (s, 2H),
5.26 (s, 2H), 6.19 (dt, J 5.9,15.7 Hz,1H), 6.69 (br s,1H), 6.74 (br s,1H),
6.88 (d, J 7.8 Hz, 2H), 7.09 (d, J 15.7 Hz, 1H), 7.30 (d, J 7.8 Hz, 2H); 13C
NMR (125 MHz, CDCl3)
d 167.7 (C), 162.2 (C), 159.2 (C), 156.7 (C),
1035, 849; 1H NMR (500 MHz, CDCl3)
d
1.27 (s,12H), 3.80 (s, 3H), 4.07
141.0 (C), 131.0 (CH), 130.3 (C), 129.6 (2ꢂCH), 129.5 (CH), 113.8
(2ꢂCH), 113.0 (C), 106.1 (CH), 101.2 (CH), 95.6 (CH2), 71.8 (CH2), 70.2
(CH2), 56.7 (CH3), 55.6 (CH3), 55.3 (CH3); HRMS calcd for
C21H24NaO7 [MþNa]þ 411.1420, found 411.1407. Next, to a mixture
of the acid in dry toluene (23 mL), triphenylphosphine (1.27 g,
(dd, J 1.5, 4.6 Hz, 2H), 4.46 (s, 2H), 5.71e5.77 (br d, 1H), 6.67 (dt, J 4.6
18.1 Hz, 1H), 6.87 (d, J 8.6 Hz, 2H), 7.27 (d, J 8.6 Hz, 2H); 13C NMR
(125 MHz, CDCl3)
d
159.1 (C), 149.3 (CH), 130.3 (C), 129.2 (2ꢂCH),
119.2 (CH), 113.7 (2ꢂCH), 83.2 (2ꢂC), 72.0 (CH2), 71.4 (CH2), 55.2
(CH3), 24.7 (4ꢂCH3). The pinacol borane 11 (1.15 g, 3.78 mmol),
Pd(PPh3)2Cl2 (483 mg, 0.688 mmol), triflate 7 (1.28 g, 3.44 mmol),
4.86 mmol), alcohol 5 (510 mg, 2.43 mmol), and DIAD (956 mL,
4.86 mmol) were added sequentially. After 30 min, EtOAc and brine
were added. The layers were separated and the aqueous phase was
extracted with EtOAc. The combined organic portions were dried
(Na2SO4), filtered, and the solvent was removed under diminished
pressure. Chromatography of the residue (petroleum ether/ace-
tone, 95:5 to 80:20) afforded 12 as a pale yellow oil; 1H NMR
and triethylamine (478 mL, 3.44 mmol) were added to a flask con-
taining n-PrOH (30 mL) and stirred whilst heating at reflux for 4.5 h.
Volatile components were evaporated under diminished pressure.
The residue was partitioned between Et2O and H2O; the layers were
separated and the organic phase was dried (Na2SO4), filtered, and the
solvent was removed under diminished pressure. Chromatography
of the residue (petroleum ether/acetone, 90:10e80:20) afforded 4 as
(500 MHz, CDCl3)
d 1.34 (d, J 6.3 Hz, 3H), 1.82e1.92 (m, 1H),
1.92e2.03 (m, 1H), 3.43 (s, 3H), 3.55 (t, J 6.5 Hz, 2H), 3.79 (s, 6H),
3.80 (s, 3H), 4.11 (br d, J 5.8 Hz, 2H), 4.39 (d, J 11.5 Hz, 1H), 4.44 (d, J
11.5 Hz, 1H), 4.45 (s, 2H), 5.10 (d, J 6.9 Hz, 1H), 5.11 (d, J 6.9 Hz, 1H),
5.32e5.40 (m, 1H), 6.26 (dt, J 5.8, 15.7 Hz, 1H), 6.59e6.68 (m, 2H),
6.69 (d, J 2.0 Hz, 1H), 6.86 (d, J 8.6 Hz, 2H), 6.87 (d, J 8.7 Hz, 2H),
a yellow oil (1.06 g, 77%); 1H NMR (500 MHz, CDCl3)
d 3.47 (s, 3H),
3.81 (s, 6H), 3.88 (s, 3H), 4.14 (d, J 5.8 Hz, 2H), 4.48 (s, 2H), 5.16 (s, 2H),
6.26 (dt, J 5.8,15.8 Hz,1H), 6.61 (d, J 15.8 Hz,1H), 6.63 (d, J 1.8 Hz,1H),
6.70 (d, J 1.8 Hz, 1H), 6.89 (d, J 8.5 Hz, 2H), 7.29 (d, J 8.5 Hz, 2H); 13C
NMR (125 MHz, CDCl3)
d
168.2 (C), 161.3 (C), 159.2 (C), 155.6 (C),
7.23e7.29 (m, 4H); 13C NMR (125 MHz, CDCl3)
d 167.2 (C), 161.1 (C),
136.9 (C), 130.2 (C), 129.7 (CH), 129.4 (2ꢂCH), 128.9 (CH), 116.7 (C),
113.8 (2ꢂCH), 103.8 (CH), 101.3 (CH), 94.9 (CH2), 71.8 (CH2), 70.2
(CH2), 56.2 (CH3), 55.5 (CH3), 55.3 (CH3), 52.2 (CH3); HRMS calcd for
C22H26NaO7 [MþNa]þ 425.1576, found 425.1588.
159.2 (C), 159.1 (C), 155.4 (C), 136.6 (C), 130.4 (C), 130.2 (C), 129.6
(CH), 129.4 (2ꢂCH), 129.2 (2ꢂCH), 128.7 (CH), 117.3 (C), 113.8
(4ꢂCH), 103.5 (CH), 101.1 (CH), 94.6 (CH2), 72.7 (CH2), 71.9 (CH2),
70.2 (CH2), 69.5 (CH), 66.5 (CH2), 56.1 (CH3), 55.5 (CH3), 55.3
(2ꢂCH3), 36.1 (CH2), 20.3 (CH3); HRMS calcd for C33H40NaO9
[MþNa]þ 603.2570, found 603.2596. Purification of 12 proved to be
difficult via chromatography. An unpurified sample of 12 was thus
subjected to the following deprotection step. To a stirred CH2Cl2/
phosphate buffer (pH 7.6) (9:1, 50 mL) emulsion containing 12,
DDQ (1.35 g, 5.95 mmol) was added in one portion at room tem-
perature. After 3 h the mixture was diluted with CH2Cl2 and
washed three times with water; the organic layer was dried
(Na2SO4), filtered, and the solvent was removed under diminished
pressure. Chromatography of the residue (petroleum ether/ace-
tone, 90:10e75:25) gave 13 as a pale yellow oil (411 mg, 50% over
6.1.3. (2R)-4-(4-Methoxybenzyloxy)-butan-2-ol 5. A mixture of al-
cohol 169 (600 mg, 2.94 mmol) and 2-(4-methoxybenzyloxy)-4-
methylquinoline 10 (1.64 g, 5.88 mmol) was placed under an at-
mosphere of argon, dissolved in CH2Cl2 (10 mL), and treated with
CSA (68.2 mg, 0.294 mmol) to afford a clear, bright yellow solution.
The mixture was stirred for 40 h at room temperature, then vola-
tiles were removed under diminished pressure. Chromatography of
the residue (petroleum ether/EtOAc, 98:2e95:5) afforded the MPM
ether intermediate as a colorless oil (876 mg, 92%); 1H NMR
(500 MHz, CDCl3)
d 0.04, 0.05 (2s, 6H), 0.88 (s, 9H), 1.13 (d, J 6.1 Hz,
3H), 1.66e1.75 (m, 2H), 3.45e3.56 (m, 2H), 3.80 (s, 3H), 3.93e4.01
(m,1H), 4.38 (d, J 11.4 Hz,1H), 4.44 (d, J 11.4 Hz,1H), 6.87 (d, J 8.6 Hz,
three steps); 1H NMR (500 MHz, CDCl3)
d 1.42 (d, J 6.3 Hz, 3H),
1.81e1.99 (m, 2H), 3.49 (s, 3H), 3.72e3.83 (m, 2H), 3.85 (s, 3H), 5.19
(d, J 7.1 Hz, 1H), 5.21 (d, J 7.1 Hz, 1H), 5.40e5.49 (m, 1H), 6.65 (dd, J
7.6, 15.8 Hz, 1H), 6.79 (d, J 1.8 Hz, 1H), 6.82 (d, J 1.8 Hz, 1H), 7.57 (d, J
2H), 7.25 (d, J 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d
159.1 (C),
130.7 (C), 129.3 (2ꢂCH), 113.7 (2ꢂCH), 72.6 (CH2), 67.0 (CH2), 65.7

5538
C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
15.8 Hz, 1H), 9.68 (d, J 7.6 Hz, 1H); 13C NMR (125 MHz, CDCl3)
193.4 (CH), 167.0 (C), 161.6 (C), 156.0 (C), 148.9 (CH), 134.1 (C), 131.1
1294, 1173, 1072; 1H NMR (500 MHz, CDCl3)
4.10 (s, 2H), 4.42e4.51 (m, 4H); 13C NMR (125 MHz, CDCl3)
d
3.34 (d, J 22.1 Hz, 2H),
195.2
d
d
(CH), 118.0 (C), 104.7 (CH), 104.0 (CH), 94.9 (CH2), 70.4 (CH), 59.0
(CH2), 56.4 (CH3), 55.7 (CH3), 38.8 (CH2), 20.5 (CH3); HRMS calcd for
C17H22NaO7 [MþNa]þ 361.1263, found 361.1267.
(d, J 7.0 Hz, C), 122.3 (dq, J 8.0, 276.0 Hz, 2ꢂCF3), 62.7 (dq, J 5.5,
37.9 Hz, 2ꢂCH2), 58.4 (d, J 5.2 Hz, CH2), 38.8 (d, J 138.1 Hz, CH2);
HRMS calcd for C7H7F6N3O4P [MꢁH]ꢁ 342.0078, found 342.0087.
6.1.5. (2S)-4-Hydroxybutan-2-yl 2-[(1E)-3-[(tert-butyldimethylsilyl)
oxy]-3-oxoprop-1-en-1-yl]-4-methoxy-6-(methoxymethoxy)benzo-
ate 14. To a solution of aldehyde 13 (388 mg, 1.15 mmol) in t-BuOH
(18 mL) was added 2-methyl-2-butene (1.22 mL, 11.5 mmol). A so-
lution of NaClO2 (207 mg, 2.29 mmol) and NaH2PO4$2H2O (1.17 g,
7.47 mmol) in water (18 mL) was then added and the mixture was
stirred overnight at room temperature. Brine was added and the
mixture was extracted three times with EtOAc. The combined or-
ganic portions were dried (Na2SO4), filtered, and volatiles were
evaporated under diminished pressure. The acid was obtained as
a yellow wax and progressed to the next step without any purifi-
cation. tert-Butylsilyl chloride (173 mg, 1.15 mmol) was added to
6.1.8. tert-Butyl 3-(bis(2,2,2-trifluoroethoxy)phosphoryl)-2-
oxopropylcarbamate 3. LHMDS (3.58 mL of 1 M in THF, 3.58 mmol)
was added dropwise to a solution of bis(2,2,2-trifluoroethyl)meth-
ylphosphonate (619 mg, 2.38 mmol) and TMEDA (537 mL,
3.58 mmol) in dry THF (6 mL), which had been pre-cooled to ꢁ98 ꢀC.
After 15 min, a solution of Boc-glycine methyl ester 18 (151 mg,
0.800 mmol) in THF (2 mL) was added and the reaction mixture was
stirred at ꢁ98 ꢀC for 100 min. The reaction mixture was then
warmed to room temperature, diluted with Et2O, washed with water
and brine, dried (Na2SO4), filtered, and concentrated under reduced
pressure. Chromatography of the residue (petroleum ether/acetone,
90:10e80:20) gave 3 as a pale yellow oil (176 mg, 53%); 1H NMR
a solution of acid and Et3N (160
m
L, 1.15 mmol) in dry THF (20 mL).
(500 MHz, CDCl3)
4.6 Hz, 2H), 4.40e4.50 (m, 4H), 5.16 (br s, 1H); 13C NMR (125 MHz,
CDCl3) 197.4 (d, J 6.6 Hz, C), 155.7 (C), 122.4 (dq, J 8.2, 276.0 Hz,
2ꢂCF3), 80.5 (C), 62.6 (dq, J 5.6, 38.0 Hz, 2ꢂCH2), 51.4 (d, J 6.0 Hz,
CH2), 38.6 (d, J 138.6 Hz, CH2), 28.2 (3ꢂCH3); HRMS calcd for
C12H17F6NO6P [MꢁH]ꢁ 416.0698, found 416.0700.
d 1.45 (s, 9H), 3.31 (d, J 21.9 Hz, 2H), 4.09 (d, J
The mixture was stirred at room temperature for 4 h, then EtOAc
was added. The organic portion was washed with water, dried
(Na2SO4), filtered, and volatiles were evaporated under reduced
pressure. Chromatography of the residue (petroleum ether/ace-
tone, 90:10) afforded 14 as a pale yellow oil (450 mg, 84% over two
d
steps); 1H NMR (500 MHz, CDCl3)
d 0.32 (s, 6H), 0.98 (s, 9H), 1.40 (d,
J 6.2 Hz, 3H), 1.77e1.87 (m, 1H), 1.88e1.98 (m, 1H), 2.60e2.69 (m,
1H), 3.48 (s, 3H), 3.67e3.83 (m, 2H), 3.84 (s, 3H), 5.17 (d, J 6.9 Hz,
1H), 5.20 (d, J 6.9 Hz, 1H), 5.38e5.47 (m, 1H), 6.35 (d, J 15.7 Hz, 1H),
6.76 (br s, 2H), 7.66 (d, J 15.7 Hz, 1H); 13C NMR (125 MHz, CDCl3)
6.1.9. (2E)-3-[2-({[(2S,4E)-7-{[(tert-Butoxy)carbonyl]amino}-6-
oxohept-4-en-2-yl]oxy}carbonyl)-5-methoxy-3-(methoxymethoxy)
phenyl]prop-2-enoic acid 24. Potassium carbonate (62.1 mg,
0.450 mmol) was added to a stirred solution of phosphonate 3
(125 mg, 0.300 mmol) in dry THF (15 mL), which had been pre-
cooled to ꢁ10 ꢀC. The resulting mixture was stirred at ꢁ10 ꢀC for
20 min, then aldehyde 15 (140 mg, 0.300 mmol) dissolved in dry
THF (2 mL) was added. The mixture was allowed to gradually reach
room temperature and stirring was prolonged overnight. 1 M aq
HCl solution was added; the layers were separated and the aqueous
phase was extracted with EtOAc. The combined organic portions
were dried (Na2SO4), filtered, and the solvent was removed under
diminished pressure. Chromatography of the residue (petroleum
ether/acetone, 70:30e0:100) gave 24 as a yellow wax (75.0 mg,
d
167.0 (C), 166.5 (C), 161.4 (C), 155.6 (C), 141.8 (CH), 134.3 (C), 122.9
(CH), 118.5 (C), 104.3 (CH), 103.3 (CH), 94.8 (CH2), 70.0 (CH), 58.6
(CH2), 56.3 (CH3), 55.6 (CH3), 38.6 (CH2), 25.6 (3ꢂCH3), 20.5 (CH3),
17.7 (C), ꢁ4.7 (2ꢂCH3); HRMS calcd for C23H36NaO8Si [MþNa]þ
491.2077, found 491.2091.
6.1.6. (2S)-4-Oxobutan-2-yl 2-[(1E)-3-[(tert-butyldimethylsilyl)oxy]-
3-oxoprop-1-en-1-yl]-4-methoxy-6-(methoxymethoxy)benzoate
15. DesseMartin periodinane (390 mg, 0.920 mmol) was added to
14 (214 mg, 0.457 mmol) stirred in dry CH2Cl2 (20 mL). The
resulting mixture was stirred at room temperature for 30 min, then
loaded onto a short column of silica gel and eluted with a mixture
of petroleum ether/acetone (90:10) to afford 15 (162 mg, 76%) as
50%); 1H NMR (500 MHz, CDCl3)
d 1.41 (d, J 6.3 Hz, 3H), 1.47 (s, 9H),
2.59e2.65 (m, 2H), 3.47 (s, 3H), 3.84 (s, 3H), 4.29 (br d, J 4.4 Hz, 2H),
5.17 (d, J 6.9 Hz, 1H), 5.20 (d, J 6.9 Hz, 1H), 5.29e5.38 (m, 1H),
5.47e5.54 (m, 1H), 6.18 (d, J 16.1 Hz, 1H), 6.37 (d, J 15.7 Hz, 1H), 6.77
(s, 2H), 6.89 (dt, J 7.0, 16.1 Hz, 1H), 7.72 (d, J 15.7 Hz, 1H); 13C NMR
a colorless oil; 1H NMR (500 MHz, CDCl3)
d 0.32 (s, 6H), 0.98 (s, 9H),
1.46 (d, J 6.3 Hz, 3H), 2.69 (ddd, J 1.3, 5.3 16.9 Hz, 1H), 2.82 (ddd, J
2.5, 7.2, 16.9 Hz, 1H), 3.46 (s, 3H), 3.83 (s, 3H), 5.16 (br s, 2H),
5.61e5.70 (m, 1H), 6.33 (d, J 15.7 Hz, 1H), 6.75 (s, 2H), 7.64 (d, J
(125 MHz, CDCl3) d 193.9 (C), 168.4 (C), 166.3 (C), 161.5 (C),156.5 (C),
155.9 (C), 143.4 (CH), 142.4 (CH), 134.1 (C), 130.5 (CH), 121.1 (CH),
118.1 (C), 104.1 (CH), 103.5 (CH), 94.8 (CH2), 80.7 (C), 70.8 (CH), 56.3
(CH3), 55.6 (CH3), 47.9 (CH2), 39.1 (CH2), 28.3 (3ꢂCH3), 20.1 (CH3);
HRMS calcd for C25H32NO10 [MꢁH]ꢁ 506.2026, found 506.2007.
15.7 Hz, 1H), 9.79e9.81 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 199.3
(CH), 166.3 (C), 166.1 (C), 161.5 (C), 155.9 (C), 141.5 (CH), 134.6 (C),
123.2 (CH), 117.6 (C), 104.3 (CH), 103.0 (CH), 94.6 (CH2), 67.2 (CH),
56.2 (CH3), 55.6 (CH3), 49.4 (CH2), 25.6 (3ꢂCH3), 20.2 (CH3), 17.7 (C),
ꢁ4.8 (2ꢂCH3); HRMS calcd for C23H35O8Si [MþH]þ 467.2101, found
467.2107. Product, which was slightly contaminated with by-
products from the periodinane was used in the subsequent step.
6.1.10. (3S,5E,11E)-16-Hydroxy-14-methoxy-3-methyl-3,4,8,9-
tetrahydro-1H-2,9-benzoxazacyclotetradecine-1,7,10-trione
1. Trifluoroacetic acid (200 mL) was added to a stirred solution of 24
(3.6 mg, 0.007 mmol) in dry CH2Cl2 (5 mL). The resulting mixture
was stirred at room temperature for 2 h, then volatile components
were evaporated under reduced pressure. The residue was dis-
solved in DMF (10 mL) and HOBT (2.0 mg, 0.015 mmol) and EDC
(2.9 mg, 0.015 mmol) were added and the resulting mixture was
stirred overnight at room temperature. EtOAc was then added, the
organic solution was washed with 1 M aq HCl solution and brine,
dried (Na2SO4), filtered, and concentrated under reduced pressure.
Chromatography of the residue (petroleum ether/acetone,
80:20e60:40) gave 1 as an amorphous white solid (0.75 mg, 30%
6.1.7. Bis(2,2,2-trifluoroethyl) 3-azido-2-oxopropylphosphonate
19. LHMDS (1.95 mL of 1 M in THF, 1.95 mmol) was added drop-
wise to a solution of bis(2,2,2-trifluoroethyl)methylphosphonate
(338 mg, 1.30 mmol) and TMEDA (295 mL, 1.95 mmol) in dry THF
(3 mL), which had been pre-cooled to ꢁ98 ꢀC. After 15 min, a solu-
tion of ester 1717 (50.0 mg, 0.433 mmol) in THF (1 mL) was added
and the reaction mixture was stirred at ꢁ98 ꢀC for 100 min. The
reaction mixture was then warmed to room temperature, diluted
with Et2O, washed with water and brine, dried (Na2SO4), filtered,
and concentrated under reduced pressure. Chromatography of the
residue (petroleum ether/EtOAc, 90:10e60:40) gave 19 as a yellow
oil (52.1 mg, 35%); IR (neat, cmꢁ1): 2976, 2922, 2113, 1732, 1418,
over two steps); [
a]
20 ꢁ51.7 (c 0.156, CHCl3); IR (neat, cmꢁ1): 3406
D
(br), 2925, 2853, 1645, 1258, 1162; 1H NMR (500 MHz, CDCl3)
d
1.43
(d, J 6.3 Hz, 3H), 2.48 (dddd, J 1.1, 4.5, 6.6, 15.2 Hz, 1H), 2.68 (ddd, J

C. Napolitano et al. / Tetrahedron 68 (2012) 5533e5540
5539
3.8, 8.2, 15.2 Hz, 1H), 3.84 (s, 3H), 4.19e4.28 (m, 2H), 5.44e5.53 (m,
1H), 6.15e6.24 (m, 1H), 6.27 (d, J 16.0 Hz, 1H), 6.31 (br d, J 16.3 Hz,
1H), 6.46 (br s,1H), 6.48 (d, J 2.5 Hz,1H), 6.77e6.87 (m,1H), 7.77 (d, J
minimizations and MD simulations were carried out using the
YASARA program.
16.0 Hz, 1H), 11.76 (br s, 1H); 13C NMR (125 MHz, CDCl3)
d 197.7 (C),
Acknowledgements
170.3 (C), 168.2 (C), 165.7 (C), 164.3 (C), 141.8 (CH), 140.4 (CH), 139.3
(C), 132.6 (CH), 124.8 (CH), 108.2 (CH), 104.1 (C), 101.5 (CH), 71.7
(CH), 55.6 (CH3), 49.0 (CH2), 38.0 (CH2), 18.9 (CH3); HRMS calcd for
C18H18NO6 [MꢁH]ꢁ 344.1134, found 344.1138.
Research described herein was funded by a Principal In-
vestigator Grant from Science Foundation Ireland (PI/IN1/B966).
We are also grateful to Dr. Daniele Lo Re for collection of some
spectroscopic data and helpful discussions.
6.2. Preparation of human ERK2 for docking and molecular
dynamics
Supplementary data
1H and 13C NMR spectra for all the new compounds described
herein and standard operating procedures (SOPs) for kinase assays
are available free of charge via the Internet. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.tet.2012.04.082.
The crystal structure of nonphosphorylated human ERK2 com-
plexed with FR148083 was retrieved from the protein data bank,
PDB ID: 2E14.26 The crystal structure has a missing activation loop
in the region from residues Pro176 to Tyr187. Homology modeling
was therefore performed for the entire human ERK2 amino acid
sequence (FASTA sequence, downloaded from NCBI protein data-
base) using the automated homology modeling module in
YASARA27 with the 2E14 crystal structure as the template. The
obtained homology model was further minimized with the AMBER
03 force field28 using YASARA, and used as a receptor for the sub-
sequent dockings. Although the homology model has 10 additional
residues toward the N-terminal compared to the 2E14 template, the
2E14 crystal structure numbering scheme has been used
throughout.
References and notes
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6.3. Docking
Models of 1 and hypothemycin were built in the molecular
operating environment (MOE) program,29 minimized with the
MMFF94x force field,30 and used as starting structures for docking.
The compounds were docked using the alpha PMI (principle mo-
ments of inertia) placement method, which is suitable for docking
ligands to tight receptor pockets, and affinity dG scoring methods.
The obtained docking results were further refined using the
MMFF94x force field refinement method. For each system, the pose
with highest possible scoring value and simultaneous proper
alignment in the active site was selected, and the docked enzy-
meeligand complexes used as starting structures for energy min-
imization and MD simulations. Hundred docked poses were
initially selected for each system. Following force field refinement,
the poses that deviated significantly or moved out of the active site
after minimization were discarded, and the ones remaining in the
active site retained.
4. Schirmer, A.; Kennedy, J.; Murli, S.; Reid, R.; Santi, D. V. Proc. Natl. Acad. Sci. U.S.
A. 2006, 103, 4234e4239.
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Mihara, M.; Tsuchiya, M.; Matsumoto, K. J. Biol. Chem. 2003, 278, 18485e18490.
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Huang, L. J. Antibiot. 1999, 52, 1086e1094.
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T.; Fransioli-Ignazio, L.; Jordi, M.; Drueckes, P.; Trappe, J.; Fabbro, D.;Altmann, K.-H.
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Winssinger, N. ChemBioChem 2010, 11, 1692e1699; (d) Du, H.; Matsushima, T.;
Spyvee, M.; Goto, M.; Shirota, H.; Gusovsky, F.; Chiba, K.; Kotake, M.; Yoneda, N.;
Eguchi, Y.; DiPietro, L.; Harmange, J.-C.; Gilbert, S.; Li, X.-Y.; Davis, H.; Jiang, Y.;
Zhang, Z.; Pelletier, R.; Wong, N.; Sakurai, H.; Yang, H.; Ito-Igarashi, H.; Kimura, A.;
Kuboi, Y.; Mizui, Y.;Tanaka, I.;Ikemori-Kawada, M.;Kawakami, Y.;Inoue,A.;Kawai,
T.; Kishi, Y.; Wang, Y. Bioorg. Med. Chem. Lett. 2009, 19, 6196e6199; (e) Goto, M.;
Chow, J.; Muramoto, K.; Chiba, K.; Yamamoto, S.; Fujita, M.; Obaishi, H.; Tai, K.;
Mizui, Y.; Tanaka, I.; Young, D.; Yang, H.; Wang, Y. J.; Shirota, H.; Gusovsky, F. J.
Pharmacol. Exp. Ther. 2009, 331, 485e495; (f) Dakas, P.-Y.; Barluenga, S.; Totzke, F.;
Zirrgiebel, U.; Winssinger, N. Angew. Chem., Int. Ed. 2007, 46, 6899e6902; (g)
Hearn, B. R.; Sundermann, K.; Cannoy, J.; Santi, D. V. ChemMedChem 2007, 2,
1598e1600.
8. Shen, Y.; Du, H.; Kotake, M.; Matsushima, T.; Goto, M.; Shirota, H.; Gusovsky, F.;
Li, X.; Jiang, Y.; Schiller, S.; Spyvee, M.; Davis, H.; Zhang, Z.; Pelletier, R.; Ike-
mori-Kawada, M.; Kawakami, Y.; Inoue, A.; Wanga, Y. Bioorg. Med. Chem. Lett.
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6.4. Molecular dynamics simulations
Each docked enzymeeligand complex was placed in a cubic box
ꢀ
of size 76ꢂ70ꢂ85 A with periodic boundary conditions and sol-
vated with approximately 5800 TIP3P31 water molecules. Counter
ions Naþ and Clꢁ were randomly placed to neutralize the cell, and
provide a physiologically relevant salt concentration of 0.9% (mass
percent). Long-range interactions were treated with the Particle
11. Mitsunobu, O. Synthesis 1981, 1e28.
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