Triphenylbutanamines: Kinesin spindle protein inhibitors with in vivo antitumor activity

Article

pubs.acs.org/jmc

Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in

Vivo Antitumor Activity^†

Fang Wang,*^,‡,∥,⊥James A. D. Good,^‡,∥Oliver Rath,^‡Hung Yi Kristal Kaan,^‡,#Oliver B. Sutcliffe,^§

Simon P. Mackay,^§and Frank Kozielski*^,‡

^‡Molecular Motor Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD,

Scotland, U.K.

^§Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street, University of Strathclyde, Glasgow G4 0RE,

Scotland, U.K.

S

* Supporting Information

ABSTRACT: The human mitotic kinesin Eg5 represents a

novel mitotic spindle target for cancer chemotherapy. We

previously identified S-trityl-^L-cysteine (STLC) and related

analogues as selective potent inhibitors of Eg5. We herein

report on the development of a series of 4,4,4-triphenylbutan-

1-amine inhibitors derived from the STLC scaffold. This new

generation systematically improves on potency: the most

potent C-trityl analogues exhibit K_i^app≤ 10 nM and GI₅₀≈ 50 nM, comparable to results from the phase II clinical benchmark

ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an

allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development

and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib

possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude

mice xenograft studies.

certain tumor cell lines by both caspase-dependent and

-independent pathways.^9,10Furthermore, the p-methoxy

analogue 5 has recently been shown to significantly prolong

survival in in vivo experiments using bladder and prostate

cancer xenografts in nude mice.^11,12As with most other

allosteric Eg5 specific inhibitors, STLC binds in an allosteric

pocket formed by helix α2/loop L5 and helix α3.^13,14Initial

structure−activity relationship (SAR) studies performed prior

to detailed structural analysis of Eg5−STLC complexes resulted

in the development of analogues with GI₅₀values in the range

of ∼200 nM (Figure 1, 2 and 3).^15,16Recent investigations have

identified novel analogues with improved potency in cell-based

assays.^17,18

INTRODUCTION

■

The mitotic kinesin Eg5 is a promising potential therapeutic

target for small molecule inhibitors and has been validated in

preclinical in vivo models of cancer to be essential for the

progression of mitosis. Eg5 is instrumental in forming the

bipolar spindle by separation of the duplicate spindle poles in

the early prometaphase stage of mitosis.^1,2Several Eg5 and

other mitotic kinesin inhibitors are at preclinical and clinical

stages (reviewed in ref 3), including ispinesib, one of the most

advanced Eg5 inhibitors currently in multiple phase II clinical

trials.⁴⁻⁶Previously, we identified S-trityl-L-cysteine (STLC) as

a potent and selective inhibitor of human Eg5 (Figure 1, 1).^7,8

We have determined previously the structure of Eg5

complexed with STLC (1) and identified the key binding inter-

actions in the inhibitor-binding pocket (Figure 2A and 2B).¹⁴

The three phenyl rings of the trityl headgroup are situated

in a predominantly hydrophobic core region of the allosteric

inhibitor-binding site, in the three pockets denoted P1−P3

(Figure 2B), while the cysteine tail forms several important

hydrogen bond interactions with Eg5, in agreement with those

observed in another Eg5−STLC complex by Kim et al.¹⁹Prior

SAR studies had demonstrated that STLC analogues with a

lipophilic substituent on one phenyl ring generally displayed

increased potency,^15,16,18which we confirmed structurally by

Figure 1. STLC and representative examples of previously reported

analogues.

STLC and its derivatives induce mitotic arrest by activation of

the mitotic checkpoint, which eventually leads to apoptosis in

Received: September 9, 2011

Published: January 16, 2012

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Figure 2. Eg5−inhibitor structures showing important hydrophobic, aromatic, and hydrogen bond (represented by broken lines) interactions

between the protein and ligands. Structural water molecules are represented by red spheres. (A) STLC (1), colored green, in the Eg5 inhibitor-

binding pocket. (B) Surface diagram showing an STLC analogue (gray) with a para-substituted chlorine on one phenyl ring, in the inhibitor-binding

pocket. The chlorine (green) sits in the hydrophobic P3 pocket. (C) Stereoplot showing 25 (blue) in the inhibitor-binding pocket. (D) Stereoplot

showing (S)-29 (pink) and (R)-29 (yellow) in the inhibitor-binding pocket.

solving the crystal structure of Eg5 with an STLC analogue

incorporating a p-chlorophenyl substituent in the hydrophobic

P3 region (Figure 2B).¹⁷Crystal structures of other ligands

bound to Eg5 have revealed that small polar substituents

like the m-hydroxy group in (S)-monastrol and its analogues

interact with residues lining the P2 pocket, such as Glu118,

Arg119, and Ala133.^20,21Crystallographic studies on STLC and

its analogues have also established that the phenyl ring in

P1 π-stacked with the Tyr 211 residue and exposed its lead-

ing edge to bulk solvent near Glu215 (Figure 2A). These data

from both structural observations and previous SAR studies

provided the foundation to investigate the optimal lipophilic

and polar substituents on the trityl system to improve potency

against Eg5.

physicochemical properties have profound influences on

absorption, distribution, and elimination and ultimately

determine drug efficacy.²²Our optimization strategy therefore

needed to consider the potential metabolic liability of the

thioether linkage to the trityl headgroup and the zwitterionic

nature of the cysteine amino acid group in 1. A parallel

approach was adopted for SAR investigations: we explored a

range of substituents on the trityl system by thioetherification

of tertiary alcohols to improve potency against Eg5 and in the

case of polar substituents act as alternatives to the zwitterionic

carboxylic acid in the cysteine tail. Second, isosteric replace-

ments of the sulfur heteroatom (N, O, CH₂) were investigated,

with the ultimate aim of transferring the optimal trityl head

from the STLC series to the sulfur-isosteric analogues.

We herein report on this new series of STLC analogues, the

triphenylbutanamines. They display strong in vitro basal Eg5

inhibitory activity (≤10 nM) and potently inhibit a number of

In any hit-to-lead optimization process, improving potency

against the target protein must be pursued in tandem with

optimizing the physicochemical properties of the ligand, as

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cancer tumor cell lines (≤100 nM) and improve on comparable

STLC derivatives. The in vitro and in vivo pharmacokinetic

properties of these analogues are promising and warrant further

development, and initial in vivo studies with one compound in

nude mice bearing a cell lung cancer explant xenograft LXFS

538 exhibited tumor stasis.

Scheme 3

RESULTS AND DISCUSSION

■

Synthesis. Novel compounds were synthesized as follows.

O-Trityl analogue 6 was prepared by coupling of N-(2-

hydroxyethyl)acetamide with trityl chloride followed by acetyl

hydrazinolysis (Supporting Information, Scheme S7). N-Trityl

analogues 7 and 8 were prepared via substitution of trityl chlo-

ride with FMOC-protected diamines (Scheme 1). Thioethers

a

Scheme 1

a

Reagents and conditions: (i) n-BuLi, CH₂Cl_2,rt, 30 min; (ii)

allyltrimethylsilane, FeCl₃, rt, 6 h; (iii) NaBH₄, conc H₂SO₄in Et₂O,

diglyme, rt, 3.5 h, then 75 °C, 1.5 h; (iv) 30% aq H₂O₂, 3 M aq NaOH,

rt, 6.5 h; (v) Dess−Martin periodinane, CH₂Cl₂, rt, 3 h; (vi)

benzylamine, TMSCN, Montmorillonite KSF clay, CH₂Cl₂, rt, 2 h;

(vii) 6 M HCl in dioxane, reflux, 48 h; (viii) propylene oxide, EtOH,

reflux, 30 min; (ix) HCOONH₄, 10% Pd/C, MeOH, 80 °C, 1 h.

ammonium formate based deprotection of the benzylamine 59

afforded C-trityl analogue 9 as a racemic mixture, in an overall yield

of 45%.²⁵This route also allowed access to racemic 29 and 30.

Triphenylbutan-1-amine analogues without the carboxylic acid,

exemplified by 10, were synthesized by conversion of the inter-

mediate alcohol 56 to the azide 60, with consequent Staudinger

reduction to afford the target compound 10 in an overall yield of

49% (Scheme 4).

a

Reagents and conditions: (i) TMSCl, CH₂Cl₂/THF (5:1), reflux, 2 h;

(ii) TrCl, NEt₃, CH₂Cl₂, rt, 1 h; (iii) piperidine (20% in MeOH), rt, 2

h; (iv) TrCl, NEt₃, DMF, rt, 3h.

were prepared from tertiary alcohols in trifluoroacetic acid

(Scheme 2)⁵⁸or as previously described with boron trifluoride

Determination of Stereochemistry. The racemic mix-

tures of the C-trityl amino acids rac-9, rac-29, and rac-32 were

separated by semipreparative chiral HPLC, and their absolute

stereochemistry was determined by application of a mod-

ification of Marfey’s method.^26,27This involved derivatization

of the racemic mixture with the chiral agent N_α-(2,4-dinitro-5-

fluorophenyl)-^L-valinamide (FDVA), followed by analysis of

the HPLC retention times of the resulting diastereomers

(Figure 3B(i)). In these diastereomers, the dinitrobenzene

moiety of FDVA is in a planar conformation with the diamino

substituents adopting a cis- or trans-arrangement relative to this

(Figure 3A(ii)).²⁷The diastereomer with the two more hydro-

phobic substituents in a cis-arrangement undergoes stronger

interactions with the reverse stationary phase and is retained for

longer than the corresponding diastereomer in the trans-

configuration, which enables a nonempirical determination of

the stereochemistry of amino acids and chiral amines.^27,28

Applying this method to the resolved enantiomers of 32, the

first diastereomer to elute was 32-1-FDVA. We therefore assigned

32-1 as the (S) configuration and 32-2 as the (R) enantiomer. As

Marfey’s method is an indirect method of determining absolute

stereochemistry, to ensure the diastereomers 32-1-FDVA and 32-

2-FDVA eluted in the order that would be expected under the

conditions employed, STLC 1 and STDC were evaluated by the

same methodology. STLC-FDVA was eluted first, as would be

expected from their relative stereochemistries.²⁷Further inves-

tigations with ^D- and L-cysteine and D- and L-phenylalanine also

proved consistent with designated assignments (Figure 3B).

Further assignments were based on the results of optical rotation

a

Scheme 2. General Route for Synthesis of Thioethers

a

Reagents and conditions: (i) n-BuLi, −78 °C, 1 h; (ii)

benzophenone, THF, −78 °C 4−6 h, then rt overnight; (iii)

cysteamine hydrochloride or ^L-cysteine, TFA, rt, 3 h.

and acetic acid.¹⁵Intermediate trityl alcohols were furnished by

organometallic mediated reduction of substituted benzophe-

nones and methyl esters or alternatively lithiated aryl bromides,

which allowed introduction of a wider range of substituents

to the trityl headgroup (Scheme 2). Compounds 3−5 were

obtained from the National Cancer Institute (NCI).

A more elaborate methodology was required for creation of

the C-trityl series, exemplified by compounds 9 and 10. To

introduce the carbon scaffold, the lithium alkoxide salt of trityl

alcohol was subjected to a mild allylation procedure employing

iron trichloride and allyltrimethylsilane (Scheme 3).²³The

alkene 55 was then oxidized by hydroboration−oxidation and

subsequent Swern oxidation to afford the aldehyde 57, which was

converted into the racemic protected α-aminonitrile intermediate

58 in a variation of the Strecker synthesis employing Mont-

morillonite KSF clay.²⁴After the nitrile was hydrolyzed, a mild

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a

Scheme 4

a

(i) MsCl, pyridine, rt, overnight; (ii) NaN₃, DMF, microwave, 175 °C, 10 min; (iii) Ph₃P, THF/H₂O (10:1), 60 °C, overnight.

conditions; hence, it may be removed from STLC on

encountering the acidic environment of the stomach if

administered orally. Using LC−HRMS, we established that

STLC was stable at pH 1.0 over 24 h (Supporting Information,

Table S1 and Figure S1). On the other hand, corresponding

N-trityl derivatives 7 and 8 not only displayed weak inhibition

of the basal ATPase activity of Eg5 compared with STLC but

also were completely hydrolyzed at pH 1.0 (t_1/2of ≤2.9 and

7.0 h, respectively). The oxygen analogue 6 was also a weak

inhibitor of Eg5 and decomposed after 16 h at pH 1.0 (t_1/2

=

2.5 h). We did not investigate further whether the lower activity

of these O-trityl and N-trityl analogues was due to their

instability in the assay medium or the reduced pK_aof the

primary amine and its consequent effect on the proportion of

molecules ionized to facilitate interaction with the protein.

A thioether functionality is a site for metabolism via

S-oxidation, and the enzyme phenylalanine monoxygenase has

recently been identified as a possible source for the conver-

sion of the mucoregulatory agents S-methyl-^L-cysteine and

S-carboxymethyl-L-cysteine to the corresponding inactive

S-oxides.²⁹An obvious isostere for sulfur that would not be

susceptible to acid hydrolysis or S-oxidation is a methylene

group. The carbon analogues 9 and 10 were prepared and

shown to be stable for 5 days at pH 1.0. Furthermore, they had

activity similar to that of STLC against Eg5, and when 9 was

resolved, (R)-9 was the more potent enantiomer. These

compounds also demonstrated a systematic improvement in

the K562 human leukemia cell based assays compared to their

sulfur counterparts 1 and 2, and (R)-9 reproduced its higher

activity in the cellular assay compared with its enantiomer.

Interestingly, in the Eg5 assay, the carbon analogue without the

carboxylic acid in the tail (10) showed improved activity over

the amphoteric form (9), a reversal of the trend observed with

sulfur-based analogues 1 and 2.

One of the key interactions observed in the cocrystallized

STLC−Eg5 structure was the hydrogen bond network between

the primary ammonium group of the cysteine tail and the

backbone carbonyl of Gly117 and the side chain of Glu116. To

establish whether this was being reproduced in the C-trityl

analogues 9 and 10, derivatives were prepared in which the

length of the amine tail was varied incrementally (Table S2).

A 20-fold and 8-fold decrease in Eg5 inhibitory activity was

observed for the propyl-1-amine and pentyl-1-amine analogues,

respectively, suggesting that the optimal network is achieved

with the four-carbon butyl-1-amine chain (10, Table S2).

Corresponding compounds were also prepared for the N-trityl

analogue 8 to ensure that the decreased length of the primary

amine tail was not the reason for the weak inhibitory activity

observed. In this case, an ethylene unit proximal to the N-trityl

unit again proved to be the optimal chain length (8, Table S2).

These chain length manipulations concur with the earlier

observations made for the alkyl chain in STLC.¹⁵

Figure 3. Stereochemical determination by Marfey’s method. (A) (i)

Structure of N_α-(2,4-dinitro-5-fluorophenyl)-L-valinamide (FDVA);

(ii) derivatised diastereoisomers 29-1-FDVA and 29-2-FDVA; (iii)

schematic representations of proposed cis- and trans-like diastereomer

conformations.²⁷(B) Retention times of 32-1-FDVA and 32-2-FDVA

and corresponding standard amino acids. The absorption peak at

∼18.50 corresponds to elution of unreacted FDVA.

and circular dichroism measurements when compared to the

assigned enantiomers.

Optimizing the Amino Acid Tail. Replacements of the

cysteine sulfur atom of STLC were investigated in order to

establish its overall effect on potency and ADME characteristics

(6−10, Table 1). A trityl group is often used as a protecting

group in organic synthesis that is removed under acidic

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Table 1. Heteroatom Analogues of STLC

compd

X

R

inhibition of basal ATPase activity, K_i^app(nM)

ligand efficiency

K562 cells, GI₅₀(nM)

1 (STLC)

S

(R)-CO₂H

135.9 20.5

245.5 51.2

2157.6 342.2

3018.5 398.5

2659.4 295.6

311.6 53.9

416.5 64.2

173.5 24.5

214.7 30.1

0.36

0.39

0.34

0.29

0.33

0.34

0.33

0.35

0.40

1452 76

2286 213

nd

2

H

6

O

H

7

NH

CH₂

(S)-CO₂H

H

nd

8

nd

rac-9

(S)-9

(R)-9

10

CO₂H

(S)-CO₂H

(R)-CO₂H

H

865 131

2065 168

776 26

577 61

a

nd = not determined.

Having established that carbon was an appropriate isostere in

alkyl substituents. Furthermore, electronic influences on the

attached phenyl ring are also important: 20 with a m-CF₃

group, which can be considered as similar in size to a methyl sub-

stituent but electron-withdrawing, is 4-fold less active than 16.

In our evaluation of all the crystal structures of STLC and its

analogues with Eg5, we observed in the P3 pocket a C−H···π

interaction between the phenyl group of STLC and the side

chain of Leu214 (Figure 2A). This interaction is mediated via

the π-electron clouds of the phenyl ring, so changes in its

distribution will affect the strength of its interaction with nearby

residues. Analysis of these results using a Craig plot sub-

stantiated the optimal meta-substituents as both hydrophobic

and electron-donating (Figure 3).³⁰

In the cellular assay, submicromolar activity was evident for

alkyl (16−18) and acetate substituents (24, 25, 27), which

demonstrated an approximate 2-fold improvement in this assay

versus the unsubstituted S-trityl benchmarks (1 and 2).

Paradoxically, 19 with an n-Pr substituent exhibited a dramatic

loss in cellular activity, probably as a result of limited aqueous

solubility (turbidimetric solubility of 3.75 μM). Introduction of

a carboxylic acid into the tail of 27 to produce 26 resulted in a

4-fold reduction in Eg5 activity, comparable to that seen with

25 and 24, but produced a much more pronounced reduction

in activity in the K562 cell line, which was a reversal of the

pattern seen with 1 and 2. Insufficient related compounds were

prepared in this series to determine if this trend was consistent.

From the comparable S-trityl inhibitors prepared, two pairs

with modified trityl moieties had better activity in the Eg5 basal

assay when prepared without the carboxyl acid (24 and 25; 26

and 27), while all modified S-trityl analogues with the carbo-

xylic acid moiety exhibited lower activity in the cellular assay

than their corresponding counterparts without the carboxylate

(13 and 14; 24 and 25; 26 and 27). Clearly, further investi-

gations are needed before definitive conclusions can be made

regarding the SAR role of the carboxylic acid with respect to

Eg5 inhibitory and cellular activity.

the tail for sulfur and the optimum length was a butyl chain, we

next evaluated triphenylbutan-1-amine analogues featuring

functional group replacements and/or modifications to the

alkyl chain, including those prepared as intermediates (55−60,

Table S3). However, all proved to be inactive. Replacement of

the primary amine with the secondary methylamine resulted in

a marked dropoff in activity, reinforcing the importance of a

three hydrogen bond network between the ammonium group

and the glutamate and glycine residues in the pocket for

efficient inhibition (S50, Table S3). The bulkier secondary

benzylamine derivatives rac-58 and rac-59 displayed further

reductions in activity. Replacement of the primary amine with a

primary alcohol (56, Table S3) abolished activity, which again

reinforces the need for three hydrogen bond donors and a role

for an ionic interaction between ligand and protein. Additional

N-trityl analogues incorporating cyclic piperazines in the amino

acid tail region displayed no notable activity in comparison to

either STLC 1 or 9 (data not shown).

Optimization of the Trityl Headgroup. The most potent

STLC derivatives prepared previously incorporated a lipophilic

para-substituent in one phenyl ring of the trityl moiety

(Figure 1). We have recently solved the structure of an

STLC derivative with a p-chloro on one phenyl ring, which, as

expected, occupied the environment of the P3 pocket formed

by Ile136, Pro137, Leu160, Leu214, and Phe239 (Figure 2B).¹⁸

A series of S-trityl thioethers were prepared to investigate this

environment further. Compounds exhibiting Eg5 inhibition

K_i^app≤ 100 nM were also evaluated in growth inhibition assays

using K562 cells.

While the o-chloro substituent of 11 significantly reduced

activity against Eg5 compared with STLC 1, a m-chloro (13)

produced an approximately 2-fold improvement in K_i^appfor

basal inhibition of Eg5 (Table 2). Further thioethers

incorporating meta-substituents of varying electronic, hydro-

phobic, and steric character were synthesized to optimize this

interaction, with alkyl groups particularly beneficial for Eg5

inhibitory activity. The stepwise improvement in activity from

16 (Me, K_i^app= 80 24 nM), 19 (n-Pr, K_i^app= 26.9 11 nM),

18 (i-Pr, K_i^app= 10.3 3.4 nM) to 17 (Et, K_i^app= 5.9 2.3

nM) illustrates that the interaction is very sensitive to steric

bulk at this position and most likely reflects an interaction

between the proximal lipophilic side chain of Leu214 and these

The positioning of the P1 ring extending toward the solvent

front and the nearby Glu215 residue afforded an additional

optimization opportunity to introduce more polar substituents

(Figure 2A). A series of analogues that incorporated polar

H-bond acceptor/donors on one phenyl ring was synthesized

to target bulk solvent interactions and the proximal acidic

residue. It was envisaged that these substituents could attenuate

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Table 2. STLC Analogues with Modified Trityl Rings

compd

X

R₁

2-Cl

R₂

inhibition of basal ATPase activity, K_i^app(nM)

ligand efficiency

K562 cells, GI₅₀(nM)

11

S

(R)-CO₂H

1783.9 384.0

377.4 38.4

89.9 18.5

297.8 60.0

293.9 61.8

80.0 23.9

5.9 2.3

0.29

0.36

0.37

0.40

0.45

0.42

0.39

0.33

0.37

0.34

0.37

0.30

0.35

0.31

0.38

0.35

0.40

0.41

0.46

0.44

0.41

0.39

0.42

0.44

nd

12

S

3-F

H

nd

13

S

3-Cl

(R)-CO₂H

2404 222

1045 42

nd

14

S

3-Cl

H

15

S

3-Br

H

16

S

3-Me

H

698 115

680 84

581 68

1760 124

nd

17

S

3-Et

H

18

S

3-i-Pr

3-n-Pr

3-CF₃

3-OMe

3-SMe

3-OCF₃

3-COMe

4-COMe

3-Cl

H

10.3 3.4

19

S

H

29.6 11.0

352.7 27.9

149.8 18.5

520.4 87.6

27.8 5.0

20

S

H

21

S

H

nd

22

S

H

1474 330

1518 164

964 82

706 47

4266 274

705 77

596 68

23

S

H

24

S

(R)-CO₂H

H

519.8 102.0

185.8 30.4

271.7 36.6

51.0 21.6

120.6 20.7

12.2 3.8

25

S

26

S

(R)-CO₂H

H

27

S

28

C

H

rac-29

(S)-29

(R)-29

30

3-Me

CO₂H

(S)-CO₂H

(R)-CO₂H

H

73

128 15

91

3

3-Me

11.1 3.9

3-Me

6.4 3.9

9

3-Me

8.8 1.8

200 16

337 28

31

4-Cl

H

16.2 3.1

rac-32

(S)-32

(R)-32

33

4-Me

CO₂H

(S)-CO₂H

(R)-CO₂H

H

7.5 1.7

96

5

4-Me

16.7 3.0

149

82

6

4-Me

5.4 1.7

4

4-Me

16.4 1.9

219 21

a

nd = not determined.

the highly lipophilic nature of the headgroup and produce

compounds with physicochemical properties (e.g., log D)

compatible with predicted favorable ADME profiles.^22,31

S-Trityl thioethers were again prepared to investigate these

potential interactions (Table 3), and only one compound, the

m-hydroxy STLC analogue 35, demonstrated any significant

improvement in basal Eg5 inhibitory activity over 1. This is

likely due to formation of hydrogen bonds with the main chain

nitrogen of Leu132 and the side chain of Arg119 in the P2

pocket, as observed in the crystal structures for dihydropyr-

imidine−Eg5 ternary complexes.^20,21The direct analogue 36,

which differed only by the absence of the carboxylic acid in the

tail, while 4-fold less potent in the basal assay, was 5-fold more

potent than 35 in the cellular assay. This difference can be

attributed to the difference in cell permeability resulting from

the presence of the carboxylic acid. Evaluation of the passive in

vitro cellular permeability by parallel artificial membrane

permeability assays (PAMPA) demonstrated 36 to have higher

the p-CH₂OH containing compound 47 had improved cellular

activity but reduced Eg5 inhibitory activity. This may suggest

that their cellular activity is being expressed through an

alternative mechanism to Eg5 inhibition. The meta-primary

amide 41, which has the carboxylic acid group in the tail, has

similar Eg5 inhibitory activity, compared to 42, but significantly

reduced cellular activity (32-fold less than 42 and 18-fold less

than STLC), probably due to a very low log D (log D_7.4= 1.13

for 41; log D_7.4= 1.41 for 42). Measurement of the cell

permeability by PAMPA assays found 42 to possess high

permeability of (P_app= (42.8 15.0) × 10⁻⁶cm s⁻¹), while 41

had no membrane diffusion detected when evaluated by this

method. Thus, while the meta-primary amide and secondary

amides of 42 and 43 in the trityl headgroup may act as a

suitable alternatives to the zwitterionic carboxylate in the

cysteine tail for enhancing the physicochemical balance of

STLC analogues, the dual presence of the trityl amide

modification and carboxylate appears to be incompatible with

effective cell penetration. To conclude, overall improvements to

both basal and cellular activity were associated with hydro-

phobic rather than hydrophilic para- or meta-substituents.

Combining the Optimized Head and Tail Moieties

into the Carbon Scaffold. After establishment of the SAR of

the trityl headgroup in thioethers and the optimal characteristics

of the tail group with isosteric butan-1-amine, the appropriate

para- or meta-substituted trityl groups were next translated into

permeability (P_app= (16.7

2.6) × 10⁻⁶cm s⁻¹), with no

permeability recorded for 35. All other compounds in this

series displayed only moderate inhibition of the basal activity of

Eg5 but generally similar cellular activity to 2, demonstrating a

wide tolerability of polar substrates. A modest improvement in

K562 cellular activity was observed in the meta-primary and

secondary amides 42 and 43, although their Eg5 inhibitory

activity was reduced 2-fold and 8-fold, respectively. Likewise,

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a

Table 3. STLC Analogues with Modified Trityl Rings

compd

R₁

R₂

inhibition of basal ATPase activity, K_i^app(nM)

ligand efficiency

K562 cells, GI₅₀(nM)

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

2-OH

3-OH

3-CN

CO₂H

H

1978.5 587.3

48.8 22.0

0.29

0.37

0.38

0.35

0.33

0.30

0.31

0.30

0.33

0.31

0.25

0.29

0.35

0.30

0.28

0.29

0.28

0.30

nd

2559 302

555 121

2128 108

2018 244

2133 135

2138 241

16749 6112

802 51

200.3 51.9

H

450.6 197.4

838.2 164.1

829.2 100.5

990.6 156.9

329.9 49.2

3-CH₂NH₂

3-CH₂NHCOMe

3-CO₂H

H

3-CONH₂

3-CONHMe

3-CONMe₂

3-SO₂Me

CO₂H

H

419.7 38.8

H

887.8 74.9

982 72

H

6055.6 1123.0

2089.6 246.6

432.4 91.2

2831 171

2559 180

2178 149

783 50

H

4-CN

H

4-CH₂OH

4-CH₂NH₂

4-CH₂NHCOMe

4-CONH₂

4-CONHMe

4-CONMe₂

4-SO₂Me

H

311.2 31.8

H

2942.4 782.8

1721.3 294.5

3228.6 447.4

2030.8 671.6

1526.7 359.7

1212.1 179.9

2594 191

2904 150

4335 341

3954 293

2911 271

2735 482

H

a

nd = not determined.

the C-trityl series (28−33, Table 2). We found a corresponding

systematic increase in activity against the target Eg5 protein

when evaluated in both basal in vitro and cellular growth

inhibition assays; an approximate 3-fold increase was evident for

comparable analogues 14 and 28 and 16 and 30 in the K562

assay. The most potent analogues prepared were racemic

mixtures of 29 and 32, which had the 2-aminobutanoic acid

tail and the C-trityl scaffold with a m- or p-methyl substituent on

one phenyl ring, respectively (EC₅₀of 73 3 and 95 5 nM).

The K_i^appand EC₅₀of the resolved enantiomers were also

determined (Table 2). The more active (R)-enantiomers (R)-29

and (R)-32 were low nanomolar inhibitors of Eg5 and have

has a calculated ligand efficiency of 0.32 based on the reported

K_i^app(1.7 0.1 nM),⁴indicating that our smaller tri-

phenylbutanamine scaffold possesses greater enhancement

potential.

Next, we examined the most potent analogues together with

selected control compounds, for the inhibition of the MT-

stimulated Eg5 ATPase activity (Table 5). This assay, with a

salt concentration of 150 mM, performed in the presence of

MTs, is closer to physiological conditions and allows K_i^appto be

determined in the presence of ∼5 nM Eg5 instead of ∼80 nM

employed for the basal assay, resulting in more reliable values,

in particular for the more potent analogues. The estimates for

K_i^appfor both the inhibition of the basal and MT-stimulated

ATPase activities are in good agreement. The most potent

compounds are ispinesib with K_i^appof 2.5 1.2 nM followed by

(R)-32 with 3.4 0.9 nM, which is further confirmed in cell-

based assays.

Finally, we investigated the cross-reactivity of rac-29 against a

kinesin panel of the five human kinesins Kif5a, Kif7, MPP1,

MKLP-2, and CENP-E by investigating their inhibition of the

basal and MT-stimulated ATPase activities. We did not observe

any significant inhibition for any of the kinesins tested (data not

shown), indicating that the selectivity previously observed for

STLC is maintained in this more potent analogue.⁸

estimates of K_i^appvalues of 6.4

3.9 and 5.4

1.7 nM,

respectively, which are close to the reported K_i^appof 2 nM for

ispinesib.⁴The corresponding butan-1-amine analogues (30 and

33) possess similarly low K_i^appestimates (8.8 1.8 and 16.4

1.9 nM, respectively), indicating that the presence of the

carboxylic acid is not essential for inhibition. On the other hand

the carboxylic acid containing (R)-29 and (R)-32 analogues are

approximately 2-fold more active in cell-based growth inhibition

assays. This reverses the trend observed with the sulfur

analogues, where removal of the carboxylic acid from the tail

of compounds with substituted trityl head groups in general

improved cellular activity.

The ligand efficiencies, which depend on protein targets,

were calculated for all the compounds based on their K_i^app

values (Tables 1−3) to enable useful comparison between com-

pounds with a range of molecular weights and activities.^32,33

The compounds without the carboxylic acid groups generally

showed better ligand efficiencies than their counterparts

with the carboxylic acid groups: compounds 29 and 32 had

ligand efficiencies of 0.4 and 0.41, whereas 30 and 33 showed

higher ligand efficiency of 0.46 and 0.44, respectively. Ispinesib

Crystal Structures of Active Compounds. The SAR

reported here confirms that a lipophilic substituent on one

phenyl ring generally improves potency, and we have

demonstrated that the sulfur in the tail group could be replaced

by methylene to yield compounds with systematically improved

activity both against Eg5 and in the K562 cell line. Here, we

report the crystal structures of Eg5 complexed with S-trityl

analogue 25 and triphenylbutanamine analogue 29 to a

resolution of 2.75 Å. Data collection and refinement statistics

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are presented in Table 4. Eg5−25 and Eg5−29 complexes

crystallized in space group P2₁2₁2₁and I2₁3 with 7 and 1

which does not produce any significant change in affinity.

Although the sulfur in STLC has been substituted by a carbon

in 29, the interactions between Eg5 and both inhibitors are

virtually identical, with the exception of the shorter C−C−C

bonds (3.9 Å) compared to the C−S−C bonds (4.5 Å).

In the complex with 25 where the carboxylic acid is absent,

only hydrogen bond interactions were observed between the

amino group and the main chain carbonyl oxygen of Gly117

and the side chain oxygen (OE1) of Glu116 (Figure 2C). The

loss of the carboxylate group not only removed a hydrogen

bond interaction but also appeared to reduce restraints on the

amine tail and increased its conformational freedom, as evident

from one molecule (chain D) where the amino group pointed

away from the side chain oxygen (OE1) of Glu116 and toward

the amino group of Arg221 instead. This compound has

equivalent potency with STLC, which suggests that the loss of

the carboxylate group and the greater flexibility of tail group

may be compensated by an improved interaction with the trityl

headgroup bearing a meta-substituent. Paradoxically, 24 (the

direct analogue of 25 with a carboxylate in the tail) has an

approximately 3-fold reduction in activity against Eg5 in the

basal in vitro assay, which suggests that the substituent in the

trityl group may influence the relative position of the tail so that

the carboxylate has a detrimental effect on binding.

Table 4. Data Collection and Refinement Statistics for

Eg5−25 and Eg5−29 Complexes

b

ab

,

Eg5−25

Eg5−29

unit cell dimensions

a (Å)

145.72

156.20

170.00

90

158.61

b (Å)

158.61

90

c (Å)

γ (deg)

space group

beamline/detector

P2₁2₁2₁

I2₁3

ID23-1/ADSC Q315R

7

I23-2/MAR225

1

molecules per asymmetric

unit

resolution range (Å)

no. of unique reflections

completeness (%)

multiplicity

30.0−2.75

98930 (14132)

97.7 (96.6)

5.4 (5.5)

30−2.75

17322 (2496)

99.8 (99.8)

5.8 (5.4)

R_sym(%)

9.4 (57.7)

13.0 (2.7)

70.75/0.33

8.3 (60.9)

12.2 (3.2)

77.98/0.27

I/σ(I)

c

Wilson B (Å²)/DPI (Å)

Refinement Statistics

R

_work/R_free(%)

22.06/28.04

19.76/24.30

With well-defined electron density maps, we were able to

determine unequivocally the location of the meta-substituted

acetate and methyl groups of 25 and 29, respectively. Both

substituents sit in the hydrophobic P3 pocket formed by Ile136,

Pro137, Leu160, Leu214, Phe239, and the salt bridge between

Glu116 and Arg221. The carbonyl oxygen of the acetate group

of 25 appeared to from a hydrogen bond with one of the side

chain amino groups (NH1) of Arg221 and the main chain

nitrogen of Ala218. The methyl of the acetate group was buried

in a largely hydrophobic pocket formed by Phe239, Leu160,

Ile136, and Leu214.

average B factors

overall

64.67

66.54

main chain/side chain

63.94/65.39

7/7/302

0.013

65.34/67.72

1/1/41

0.012

no. of. ADP/inhibitor/water

d

rmsd in bond length (Å)

d

rmsd in bond angle (deg)

1.70

1.75

b

a

The racemic mixtures were used for crystallization. Values in

c

parentheses pertain to the highest resolution shell. DPI: diffraction-

component precision index.⁵⁵rmsd is the root-mean-square deviation

d

from ideal geometry.

Overall, the crystal structures of Eg5−25 and Eg5−29

provide insight into the arrangement of the substituents within

the binding site and help explain some of the structure−activity

relationships of the ligands. For example, replacement of the

m-acetyl with a m-ethyl (17) or m-isopropyl (18) group

significantly improved activity by approximately 20-fold, which

could be attributed to the replacement of a polar carbonyl

group in an essentially hydrophobic pocket with the more

lipophilic alkyl group and facilitating a favorable interaction

with the side chain of Leu214. Furthermore, the electronic

properties of the phenyl ring will be altered by an alkyl (+I)

compared with an acetyl (−I), which would impact on the

strength of the π−CH interaction with Leu214. The generally

lower activities of the m-hydrophilic group series (Table 3)

compared with 25 provide further evidence that this hydro-

phobic pocket has a low tolerance for polar groups, despite

the proximity of the Glu116−Arg221 salt bridge. Shifting the

m-acetyl to the p-position (compound 27) produced a 4-fold

increase in activity, which could be due to an improved

alignment of the carbonyl with the side chain of Arg221.

Evaluation of Inhibitors across Multiple Cell Lines. Of

the 47 compounds tested in proliferation assays using human

K562 leukemia cells (Tables 1−3), the most potent were

subsequently investigated in four additional human cell lines

(Table 5). These were derived from colon, lung, and pancreatic

cancer, as well as immortalized cells derived from normal breast

tissue. From our previous study, we included three potent

S-trityl analogues (3−5) and STLC 1 as controls and to monitor

molecule per asymmetric unit, respectively. In the Eg5−25

complex, 6 out of 7 of the Eg5 molecules were in the final

inhibitor-bound state, whereby loop L5 has swung downward

to close the inhibitor-binding pocket, the switch II cluster has

moved upward, and the neck-linker has adopted a docked

conformation. The seventh molecule (chain G) was trapped in

an intermediate state similar to that in other Eg5−inhibitor

structures, whereby local changes at the inhibitor-binding

pocket have not propagated to complete structural changes at

the switch II cluster and the neck-linker remains undocked.^14,18

In the Eg5−29 complex, Eg5 was in the final inhibitor-bound

state.

Examination of both complexes revealed that the three

phenyl rings of 25 (Figure 2C) and 29 (Figure 2D) were buried

in the same pockets as STLC, with the same hydrophobic and

aromatic interactions described previously.¹⁴As we used the

racemic mixture of 29 to crystallize the Eg5−inhibitor complex,

we observed electron density for both enantiomers in the

structure. Several hydrogen bond interactions between Eg5 and

the cysteine moiety of (S)-29 were apparent: the amino group

with the main chain carbonyl oxygen of Gly117 and the side

chain oxygen (OE1) of Glu116, and an oxygen (OXT) of the

carboxylic group of the cysteine moiety with one of the side

chain amino groups (NH1) of Arg221 (Figure 2D). These

interactions were not observed for the (R)-enantiomer. Instead,

the amino group of the cysteine moiety interacts with the

protein via hydrogen bonding with a structural water molecule,

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Figure 4. Craig plot for aromatic substituents of σ vs π. π is the substituent hydrophobicity constant and σ the Hammett substitution constant, a

measure of the electronic influence of the substituent. Values were obtained from model systems from refs 30, 56, and 57.

Table 5. Testing of a Series of Representative STLC Analogues for the Inhibition of the MT-Stimulated ATPase Activity and in

Human Leukemia (K562), Lung (NCI-H1299), Pancreas (BxPC3), and Colon (HCT116) Tumor Cell Lines and a Normal

Breast Cell Line (hTERT-HME1)

GI₅₀(nM)

compd

inhibition MT-stimulated ATPase activity, K_i^app(nM)

HCT116

K562

hTERT-HME1

NCI-H1299

BxPC-3

1 (STLC)

143.8 21.6

152.3 15.0

13.6 2.4

4.2 2.3

553 57

1213 40

130 11

153 10

1452 76

2286 213

242 22

247 38

240 17

865 131

2065 168

776 26

577 61

361 75

1607 199

57 15

1549 111

2301 511

242 28

1563 155

2463 908

284 32

2

3 (NSC123139)

4 (NSC123529)

5 (NSC123528)

rac-9

119

3

227 28

245 43

9.1 2.2

130

7

81 12

294 24

291 54

235.4 47.5

384.4 61.3

211.3 24.6

203.0 44.5

5.1 3.3

828 29

1603 127

472 20

213 18

494 46

721 99

321 28

572 92

2825 219

4018 303

1324 55

964 214

1489 111

4864 545

1119 138

1225 296

(S)-9

(R)-9

10

rac-29

99

51

95

5

2

7

5

91

9

38

26

42

7

3

5

137

132

8

7

144

6

(R)-29

10.2 2.0

12.8 1.6

10.8 1.8

10.7 3.6

3.4 1.6

98 12

75 13

(S)-29

128 15

200 16

337 28

281 26

360 73

828 37

180 35

1211 265

2312 418

85 12

30

150 32

308 54

31

230 27

(R)-32

40

2

6

3

82

4

27

49

44

3

4

111

6

(S)-32

19.3 6.4

9.8 1.3

115

84

6

7

149

98

6

305 30

204 19

376 37

82 10

268 27

151 29

1603 168

80 15

rac-32

5

33

9.7 1.8

154

25

233 21

71

601 107

32

54 (ispinesib)

2.5 1.2

8

progress and the clinical phase II candidate ispinesib to benchmark

the new analogues.^4,5,15(R)-32 improves at least 2-fold on our

previously reported most potent STLC analogues (3−5) in the

selected cell lines and has comparable activity with ispinesib across

all cell lines. (R)-29, which differs from (R)-32 only by the methyl

group being in the para-position, shows only slightly lower activity

in the lung cell line. The most potent analogue lacking the

carboxylic acid group is 30. Although significant differences in

activity were not observed between S-trityl-^L-cysteine and S-trityl-

D-cysteine in previous studies, detectable differences were evident

among the enantiomers of the 2-aminobutanoic acid series.^8,18

When the sulfur of STLC was replaced with carbon to produce

(S)-9, there was lower activity across all cell lines, while the

corresponding carbon analogue of STDC [(R)-9] had comparable

activity. It is only when substituents were introduced into the

headgroup of the tritylbutanamines that significant improvements

in activity became apparent.

ADME Profiling of the Triphenylbutanamines and in

Vivo Xenograft Studies. Our two most potent compounds

with and without the carboxylic acid (29 and 30, respectively)

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Table 6. ADME Profiling of Triphenylbutanamines and Ispinesib

assay/compd

54 (ispinesib)

30

(S)-29

(R)-29

MW (Da)

517.06

315.45

359.46

a

turbidimetric solubility pH 2.0, 6.0, 7.4 (μM)

65, 65, 65

4.75 0.04

9.34 0.08

>100, >100, >100

4.69 0.05

>100, >100, >100

a

log P

pK_a

2.45 0.07

a

10.09 0.02

pK_a1: 9.07 0.03

pK_a2: 2.55 0.07

a

log D_7.4

2.66 0.24

2.33 0.16

2.45 0.19

microsomal stability [Cl_int[(μL/min)/mg protein]

human

stable

mice

stable

45.0 3.09

3.93 2.48

6.48 1.63

0.0697 0.0178

69.1

stable

human hepatocytes [(μL/min)/million cells]

hERG (μM)

0.75 2.35

4.81 1.36

0.0835 0.0023

50.6

stable

>25

human plasma protein binding (fu_100%) (% recovery)

0.191 0.0028

52.3

0.168 0.0012

50.1

CYP450 inhibition (μM)

1A2

>25

2C9

>25

4.3 0.5

3.2 0.5

8.5 0.8

5.5 0.8

4

2C19

24.3 6.5

>25

2D6

3A4

4.1 0.4

45

a

bioavailability (%)

51

a

The racemic mixture was used.

together with ispinesib to provide a benchmark were assessed

in a series of in vitro and in vivo ADME assays (Table 6; for 29,

the separated enantiomers were used for profiling). Ispinesib

had the lowest turbidimetric solubility (65 μM, partially soluble),

whereas both 30 and rac-29 were soluble (>100 μM). Despite

the nonoptimal log P of 30 and ispinesib, the log D at pH 7.4

was <3 and favorable for all compounds.^22,31During the time

course of the experiments, all inhibitors either proved stable or

exhibited low clearance in human microsomal and hepatocyte

stability assays. In mouse microsomal assays, ispinesib and both

enantiomers of 29 were stable whereas 30 showed high

microsomal clearance, indicating species-dependent differences

between mouse and human for this compound. Consequently,

the high clearance of 30 excluded it from in vivo mouse xeno-

graft studies. A further example of the need to balance efficacy

with structural modifications to improve potency is demon-

strated by hERG inhibition. Both ispinesib and 30 were

moderately potent hERG inhibitors with IC₅₀of 4.7 1.8 and

6.5 1.6 μM, respectively. Among the most successful reported

approaches for diminishing binding to the hERG channel are

modulation of lipophilicity and structural modifications that

disrupt the π-stacking and hydrophobic interactions between

the drug candidate and the channel cavity.³⁴It is therefore

not unexpected that installation of a polar, carboxylic acid

functionality into 30 to produce 29 results in a dramatic

reduction in hERG binding. Plasma protein binding influences

the distribution and elimination of compounds: the fraction

unbound of all compounds tested was >90% with (S)-29 being

slightly less bound than the other compounds and is acceptable

for this program. We also tested the compounds for inhibition

of the main CYP isoforms. (S)-29 and (R)-29 did not show

any inhibition up to 25 μM, but ispinesib moderately to

significantly inhibited isoform CYP3A4 in vitro with an IC₅₀of

4.05 0.44 μM, in agreement with two recent studies.^35,36The

four isoforms CYP2C9, CYP2C19, CYP2D6, and CYP3A4

were inhibited by 30 with moderate to significant IC₅₀between

3.17 0.45 and 8.46 0.84 μM.

We subsequently determined the bioavailability for 30, rac-

29, and ispinesib (Table 6). Compound 30 had particularly low

bioavailability of 4% and low po levels, which confirms the in

vitro high microsomal clearance values seen in mice. Ispinesib

shows moderate clearance with good po levels and has a

moderate bioavailability of 45%. Finally, rac-29 showed low

clearance, good po levels, and a bioavailability of 51%. On the

basis of rac-29 having the better ADME profile compared with

30, particularly with respect to bioavailability, we tested it in

vivo using lung cancer patient explants (LXFS 538) passaged as

subcutaneous xenografts in nude mice.

In this model, by transplantation of a tumor from a patient to

a mouse, many of the characteristics of the parental patient

tumors including histology and sensitivity to anticancer drugs

are retained. Moreover, earlier studies have shown that such

explants correctly replicate the response of the donor patient to

standard anticancer drugs in >90% of the cases.³⁷

Rac-29 displayed good antitumor activity in LXFS 538: a

minimum T/C of 26.3% was recorded, corresponding to transient

partial tumor remission (i.e., individual relative tumor volumes of

<100%) in four out of five tumors around day 10 and an obvious

reduction of growth rates compared to the vehicle control group

in the latter part of the experiment (Figure 5). This resulted in an

increase of tumor volume doubling times from 8.8 days in the

control group to 28.7 days in the rac-29-treated group. The results

became statistically significant on day 28.

CONCLUSION

■

Triphenylbutan-1-amines represent a potent class of Eg5

inhibitors, which demonstrate good in vivo antitumor activity

against lung cancer xenografts in mouse models. The SAR

modifications of meta or para lipophilic trityl substituents,

isosteric replacement of the sulfur with methylene, and

inversion of the amino acid stereocenter with respect to

STLC have produced analogues that systematically improve on

the comparable S-trityl thioethanamines. With very potent

nanomolar inhibitory activity exhibited against the target

kinesin Eg5, favorable ligand efficiencies, and similar GI₅₀

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compounds were named according to IUPAC nomenclature using ACD

ChemSketch 12.01 (Windows, Advanced Chemistry Development,

Toronto, Canada). Detailed synthetic procedures and characterization

for all other compounds are available in the Supporting Information.

1,1′,1″-But-1-ene-4,4,4-triyltribenzene (55). The title com-

pound was prepared using an adaptation of the method reported by

Kabalka et al.²³A solution of triphenylmethanol (7.81 g, 30.0 mmol)

in anhydrous CH₂Cl₂(150 mL) was treated with n-butyllithium

(1.6 M in hexane, 20.0 mL, 32.0 mmol) at 0 °C and the mixture then

warmed to room temperature. After the mixture was stirred for 30 min,

allyltrimethylsilane (5.75 mL, 36.0 mmol) and iron trichloride (5.19 g,

32.0 mmol) were added. The mixture was allowed to stir for a further

6 h and then quenched with water (40 mL). The reaction mixture was

extracted with EtOAc (100 mL), dried (MgSO₄), and concentrated

in vacuo. The crude product was purified by flash chromatography

Figure 5. Anticancer efficacy of rac-29 in a subcutaneous xenograft

tumor model. The rac-29 treatment group ( ) received 20 mg/kg on

◆

days 0, 2, and 4 and 15 mg/kg rac-29 on days 17, 19, 24 (indicated by

1

[SiO₂, hexane] to give 55 as a yellow syrup (7.9 g, 93%). H NMR

□

arrows), whereas the control group ( ) received only vehicle on the

(400 MHz, CDCl₃) δ = 3.46 (d, 2H, CH₂), 4.96−5.06 (m, 2H, CH₂),

5.64−5.68 (m, 1H, CH), 7.10−7.27 (m, 15H, Ph). ¹³C NMR (100

MHz, CDCl₃) δ = 45.6, 56.4, 117.4, 126.1, 127.9, 129.5, 147.4, 136.1.

HRMS (ESI+) calcd for C₂₂H₂₀(M + H)⁺, 285.163 78; found,

285.163 60. Anal. Calcd for C₂₂H₂₀: C, 92.91; H, 7.09. Found: C,

92.98; H, 7.27.

same days. The data are plotted as the mean of the relative tumor

volume standard deviation. The difference between treated group

and vehicle is statistically significant (p = 0.016).

values over five tumor cell lines compared to the phase II

candidate of equivalent potency, they represent excellent

compounds for continued development and optimization.

The major effect of the α-carboxylic acid is in determining

the cellular permeability, and its presence in the amine tail

reduces log P, enhances bioavailability, and attenuates inter-

actions with hERG and several CYP isoforms. The continued

development and further studies on this scaffold will be

reported in due course.

4,4,4-Triphenyl-1-butanol (56). The title compound was

prepared using an adaptation of the method reported by Starnes.³⁸

A solution of concentrated sulfuric acid (15.3 μL, 0.27 mmol) in

anhydrous diethyl ether (0.37 mL) was added dropwise (at room

temperature) to a solution of sodium borohydride (20.6 mg, 0.54

mmol) and 1,1′,1″-but-1-ene-4,4,4-triyltribenzene 55 (405 mg, 1.42

mmol) in anhydrous diglyme (3 mL). The mixture was stirred at room

temperature for 3.5 h and then heated at 75 °C for a further 1.5 h. The

mixture was cooled to 0 °C and treated successively with water

(39 μL), aqueous NaOH (3M, 0.18 mL), and 30% aqueous hydrogen

peroxide (0.18 mL). The mixture was stirred for 20 min at 0 °C and

then 6.5 h at room temperature. The mixture was extracted with Et₂O

(50 mL), washed with water (25 mL), dried (MgSO₄), and concen-

trated in vacuo. The crude product was purified by flash chromato-

graphy [SiO₂, 10−40% EtOAc in hexane] to afford the alcohol 56 as a

white solid (305 mg, 71%). Mp 118 °C (lit. 120−122 °C).³⁹¹H NMR

(400 MHz, DMSO-d₆) δ = 1.07−1.11 (m, 2H, CH₂), 2.54−2.58

(m, 2H, CH₂), 3.37−3.39 (m, 2H, CH₂), 4.46 (t, 1H, OH), 7.22−7.28

(m, 15H, Ph). ¹³C NMR (100 MHz, DMSO-d₆) δ = 29.0, 36.0, 55.9,

60.9, 125.6, 127.8, 128.7, 147.2. HRMS (ESI+) calcd for C₂₂H₂₂O (M

+ NH₄)⁺. 320.200 89; found, 320.200 58. Anal. Calcd for C₂₂H₂₂O: C,

87.38; H, 7.33. Found: C, 85.27; H, 7.54.

EXPERIMENTAL SECTION

■

(A) Chemistry. All reagents and solvents were of commercial

quality and used without further purification. STLC 1 was purchased

from Nova Biochem and used without further purification. Ispinesib

was a gift from Sanofi-Aventis. Compounds 3−5 and STDC

(NSC123139, NSC123528, NSC123529, and NSC124767) were

obtained from the NCI/DTP Open Chemical Repository (http://

dtp.cancer.gov), National Cancer Institute. Anhydrous reactions were

carried out in oven-dried glassware under a nitrogen atmosphere

unless otherwise noted. Microwave reactions were performed using a

Biotage Initiator-8 microwave synthesizer (operating at 2.45 GHz).

Thin-layer chromatography (TLC) was carried out on aluminum-

backed SiO₂plates (silica gel 60, F₂₅₄), and spots were visualized using

ultraviolet light (254 nm) or by staining with phosphomolybdic

acid. Flash column chromatography was performed on silica gel [60 Å,

35−70 μm or SNAP KP-Sil, 60 Å, 40−63 μm cartridges] manually or

using a Biotage SP4 automated chromatography system (detection

4,4,4-Triphenylbutanal (57). The title compound was prepared

using an adaptation of the procedure reported by Rodriguez et al.⁴⁰

Dess−Martin periodinane (356 mg, 0.84 mmol) was added to a stirred

solution of 4,4,4-triphenyl-1-butanol 55 (212 mg, 0.7 mmol) in

CH₂Cl₂(8 mL). The mixture was stirred at room temperature for

3 h and then quenched with saturated aqueous Na₂S₂O₃solution

(5 mL) followed by saturated aqueous NaHCO₃solution (5 mL).

The mixture was extracted with CH₂Cl₂(50 mL), dried (MgSO₄), and

concentrated in vacuo. The crude product was purified by flash

chromatography [SiO₂, 2−15% EtOAc in hexane] to give 57 as a white

1

wavelength, 254 nm; monitoring, 280 nm). H and ¹³C NMR spectra

were recorded on a JEOL ECX-400 (400 MHz), Avance DPX400

(400 MHz), or Avance DPX500 (500 MHz) spectrometer. ¹⁹F NMR

spectra were recorded on an Avance AV400 (400 MHz) instrument

equipped with a multinuclear probe. ¹H chemical shifts (δ) are

reported in ppm relative to the residual signal of the deuterated solvent

(7.26 in CDCl₃, 3.31 in CD₃OD, and 2.50 in DMSO-d₆). Multiplicities

are indicated by s (singlet), d (doublet), t (triplet), q (quartet),

p (pentet), m (multiplet), and br (broad). ¹³C chemical shifts (δ) are

reported in ppm relative to the carbon resonance of the deuterated

solvent (77.16 in CDCl₃, 49.00 in CD₃OD, and 39.52 in DMSO-d₆).

¹⁹F spectra are referenced relative to CFCl₃. Mass spectra were

recorded on a Thermo Electron LTQ ORBITRAP mass spectrometer

using electrospray ionization. Data from gas chromatography−mass

spectrometry (GC−MS) were recorded on a Thermo Scientific Focus

GC with DSQ2 single quadrupole mass spectrometer. Melting points

were determined using a Stuart Scientific SMP1 melting point

apparatus and are uncorrected. Elemental analysis data were recorded

on a Perkin-Elmer 2400 series 2 CHN analyzer. All tested compounds

were in general ≥95% pure. HPLC analysis was carried out using a

Gilson HPLC system and DIONEX P 680 HPLC system. New

1

solid (187 mg, 89%). Mp 108 °C. H NMR (400 MHz, DMSO-d₆)

δ = 2.14−2.18 (t, 2H, CH₂), 2.85−2.89 (t, 2H, CH₂), 7.23−7.29 (m,

15H, Ph), 9.47 (s, 1H, CHO). ¹³C NMR (100 MHz, DMSO-d₆) δ =

31.4, 38.9, 55.6, 125.7, 127.8, 128.7, 146.5, 202.3. HRMS (ESI+) calcd

for C₂₂H₂₀O (M + NH₄)⁺, 318.185 24; found, 318.184 81. Anal. Calcd

for C₂₂H₂₀O: C, 87.96; H, 6.71. Found: C, 87.99; H, 6.76.

2-(Benzylamino)-5,5,5-triphenylpentanenitrile (rac-58). The

title compound was prepared using an adaptation of the method

reported by Yadav et al.²⁴Montmorillonite KSF clay (1.2 g) was added

to a solution of 4,4,4-triphenylbutanal 57 (300 mg, 1.0 mmol),

benzylamine (129 mg, 132 μL, 1.2 mmol), and trimethylsilyl cyanide

(144 mg, 192 μL, 1.4 mmol) in CH₂Cl₂(12 mL). The mixture was

stirred at room temperature for 2 h, filtered and the clay rinsed with

CH₂Cl₂(15 mL). The combined organic layers were dried (MgSO₄),

concentrated in vacuo and the crude product was purified by flash

chromatography [SiO₂, 0−15% EtOAc in hexane] to give rac-58 as a

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1

white solid (388 mg, 93%). H NMR (400 MHz, DMSO-d₆) δ =

MeOH in CH₂Cl₂with 0.5% NH₄OH] to give the title compound 10

as a white solid (117 mg, 85%). Mp 72 °C. H NMR (400 MHz,

1

1.35−1.41 (m, 2H, CH₂), 2.67−2.78 (m, 2H, CH₂), 3.19−3.23 (m,

1H, NH), 3.59−3.64 (m, 2H, PhCH₂), 3.79 (dd, 1H, CH), 7.23−7.30

(m, 20H, Ph). ¹³C NMR (100 MHz, DMSO-d₆) δ = 29.2, 35.4, 49.5,

50.7, 55.8, 120.7, 125.9, 127.6, 128.6, 128.8, 146.7. HRMS (ESI+)

calcd for C₃₀H₂₈N₂(M + H)⁺, 417.232 53; found, 417.232 17. Anal.

Calcd for C₃₀H₂₈N₂·2H₂O: C, 79.61; H, 7.13; N, 6.19. Found: C,

79.03; H, 6.79; N, 5.50.

DMSO-d₆) δ = 1.02 (m, 2H, CH₂), 2.49−2.57 (m, 4H, CH₂and

CH₂), 7.22−7.40 (m, 15H, Ph). ¹³C NMR (100 MHz, DMSO-d₆) δ =

29.9, 37.5, 42.4, 56.6, 126.3, 128.4, 129.4, 147.9. HRMS (ESI+) calcd

for C₂₂H₂₃N (M + H)⁺, 302.190 33; found, 302.189 91. Anal. Calcd for

C₂₂H₂₃N·0.5H₂O: C, 85.12; H, 7.79; N, 4.51. Found: C, 85.23; H,

7.69; N, 4.46.

Enantiomeric Separation by Semipreparative Chiral Chro-

matography. Separation was performed on a Dionex P680 HPLC

system using a 10 mm × 250 mm ChiralPack IC column containing

cellulose tris(3,5-dichlorophenylcarbamate) immobilized on 5 μm

silica gel as the chiral stationary phase. Mobile phase was n-heptane/

ethanol/trifluoroacetic acid/triethylamine [95:5:0.1:0.1% v/v]. The

flow rate was 4 mL/min, and detection wavelength (UV) was 254 nm.

The separated enantiomers were impure with TFA and NEt₃.

Purification by flash chromatography [SiO₂, 0−24% MeOH in

CH₂Cl₂with 1% NH₄OH] afforded the pure enantiomers as white

solids (∼5 mg each). Retention times for compound 9: 30 and 36 min.

For 29: 32 and 40 min. For 32: 33 and 40 min.

Application of Marfey’s Method To Determine Absolute

Stereochemistry. The following general procedure was used for

derivatization of 32-1 and 32-2.⁴⁴N-α-(2,4- Dinitro-5-fluorophenyl)-L-

valinamide (1% w/v in acetone, 100 μL, 3.6 μmol) was added to a

small vial containing a solution of the amino acid (2.5 μmol) in

methanol (50 μL), followed by NaHCO₃(1 M in H₂O, 20 μL, 20

μmol), and the mixture was heated at 40 °C for 1 h with stirring. After

cooling to room temperature, the mixture was quenched with aqueous

HCl (1 M, 20 μL), diluted to 1 mL with MeOH, and then measured

by LC−MS. HPLC conditions: Dionex Ultimate 3000 system,

acetonitrile (0.1% formic acid) and water (0.1% formic acid), 15−

100% acetonitrile in 35 min. Flow rate: 0.3 mL/min. Wavelength:

254 nm.

(B) Biology. Inhibition of the Basal and MT-Stimulated Eg5

ATPase Activities. For ATPase assays and structure determination,

Eg5₁₋₃₆₈was cloned, expressed, and purified as previously described.¹⁴

The inhibition of the Eg5 ATPase activity was determined as

previously described.¹⁸In short, ATPase rates were measured using

the pyruvate kinase/lactate dehydrogenase-linked assay. The measure-

ments were performed at 25 °C in 96-well μclear plates (340 nm)

using a 96-well Tecan Sunrise photometer. The salt concentration was

150 mM NaCl for both the inhibition of the basal and the MT-

stimulated ATPase activity with an Eg5 concentration of ∼80 and

∼5 nM, respectively. It is noteworthy that for this Eg5 construct the

IC₅₀or K_i^appvalues can depend on the ionic strength of the buffer.⁴⁵

All data were measured at least in triplicate. To obtain estimates for

the apparent K_i, data were fitted to the Morrison equation:⁴⁶

2-(Benzylamino)-5,5,5-triphenylpentanoic Acid (rac-59). The

title compound was prepared using an adaptation of the procedures

reported by Bigge et al. and Warmuth et al.^41,42A solution of 2-

(benzylamino)-5,5,5-triphenylpentanenitrile rac-58 (130 mg, 0.31

mmol) in HCl (6 M in dioxane, 8 mL) was heated at reflux for 2

days and then concentrated in vacuo. The residue was redissolved in

EtOH (3 mL), propylene oxide (1 mL) added, and the mixture heated

at reflux for 30 min. The volatiles were removed in vacuo and the

crude product was purified by flash chromatography [SiO₂, 0−18%

MeOH in CH₂Cl₂with 0.5% NH₄OH] to give the protected amino

1

acid rac-59 as a white solid (118 mg, 87%). H NMR (400 MHz,

CD₃OD) δ = 1.57−1.64 (m, 2H, CH₂), 2.70−2.84 (m, 2H, CH₂), 3.30

(m, 2H, PhCH₂), 3.39−3.43 (m, 1H, NH), 3.89−4.04 (dd, 1H, CH),

7.23−7.37 (m, 20H, Ph). ¹³C NMR (100 MHz, CD₃OD) δ = 26.6,

35.4, 50.0, 56.1, 62.0, 125.7, 127.6, 128.8, 129.0, 147.0. HRMS (ESI+)

calcd for C₃₀H₂₉NO₂(M + H)⁺: 436.227 11; found, 436.227 20. Anal.

Calcd for C₃₀H₂₉NO₂: C, 82.73; H, 6.71; N, 3.22. Found: C, 82.82; H,

6.93; N, 2.52.

2-Amino-5,5,5-triphenylpentanoic Acid (rac-9). The title

compound was prepared using an adaptation of the method reported

by Siya Ram et al.²⁵A mixture of 2-(benzylamino)-5,5,5-

triphenylpentanoic acid rac-58 (189 mg, 0.43 mmol), 10% Pd/C

(94.5 mg), and HCOONH₄(137 mg, 2.17 mmol) in anhydrous

MeOH (15 mL) was heated at 80 °C for 1 h. The mixture was cooled

and then filtered through Celite, which was then washed with MeOH

(10 mL). The filtrate was concentrated in vacuo and the crude product

purified by flash chromatography [SiO₂, 5−25% MeOH in CH₂Cl₂

with 0.5% NH₄OH] to give rac-9 as a white solid (133 mg, 89%).

1

Mp 176 °C. H NMR (500 MHz, DMSO-d₆) δ = 1.26−1.45 (m,

2H, CH₂), 2.60−2.77 (m, 2H, CH₂), 3.16 (m, 1H, CH), 7.15−7.28

(m, 15H, Ph). ¹³C NMR (125 MHz, DMSO-d₆) δ = 27.7, 36.1, 55.1,

56.4, 126.2, 128.3, 129.4, 147.5, 170.5. HRMS (ESI+) calcd for

C₂₃H₂₃NO₂(M + H)⁺, 346.180 16; found, 346.179 87. Anal. Calcd

for C₂₃H₂₃NO₂: C, 79.97; H, 6.71; N, 4.05. Found: C, 79.17; H, 7.09;

N, 4.64.

1,1′,1″-(4-Azidobutane-1,1,1-triyl)tribenzene (60). Methane-

sulfonyl chloride (85 μL, 1.1 mmol) was added to a solution of 56

(166 mg, 0.55 mmol) in anhydrous pyridine (5 mL) at 0 °C (ice−

water). The mixture was stirred for 12 h at room temperature, and the

volatiles were removed in vacuo. The residue was extracted with

CH₂Cl₂(30 mL), dried (MgSO₄), and concentrated in vacuo. The

crude product was purified by flash chromatography [SiO₂, 2−30%

EtOAc in hexane] to give 4,4,4-triphenylbutylmethanesulfonate as a

white solid (199 mg, 95%). Sodium azide (130 mg, 2.0 mmol) was

added to a solution of 4,4,4-triphenylbutyl methanesulfonate (190 mg,

0.5 mmol) in anhydrous DMF (2 mL) and the mixture irradiated with

microwave radiation at 175 °C. The mixture was cooled. The solid

residue was filtered off, and the filtrate was concentrated in vacuo. The

crude product was purified by flash chromatography [SiO₂, 2−30%

EtOAc in hexane] to give the azide 60 as a white solid (150 mg, 92%).

⎧

⎡

v

i

app

⎨

= 1 − (ν

− ν ) ([E] + [I] + K

)

⎢

⎣

max

min

i

(

v

⎩

0

⎫

⎬

⎭

⎤

app 2

−

([E] + [I] + K ) − 4[E][I]

+ ν

⎥

⎦

i

min

)

/(2[E])

whereby v_i/v₀is the fractional activity, v_maxis the uninhibited protein

activity, v_minis the remaining activity at the highest inhibitor

concentration used, [E] and [I] represent the enzyme and inhibitor

concentrations used in the assays, and K_i^appis the apparent K_i

determined from the data.

Calculation of Ligand Efficiencies. The equation for calculating

ligand efficiencies is LE = −ΔG/HAC ≈ −RT ln(K_i^app)/HAC, where

ΔG is the change in Gibbs free energy, T is the absolute temperature,

R represents the gas constant, and HAC is the heavy atom count for

non-hydrogen atoms.

Tissue Culture. HCT116 (ATCC CCL-247) cells were cultured in

DMEM (Invitrogen, Paisley, U.K.), supplemented with 10% fetal

bovine serum (PAA, Pasching, Austria). K562 (ATCC CCL-243),

LNCaP (ATCC CRL-1740), and NCI-H1299 (CRL-5803) cells were

1

Mp 114 °C. H NMR (400 MHz, CDCl₃) δ = 1.37 (m, 2H, CH₂),

2.64 (m, 2H, CH₂), 3.24 (t, 2H, CH₂), 7.10−7.27 (m, 15H, Ph). ¹³C

NMR (100 MHz, CDCl₃) δ = 25.6, 37.4, 52.1, 56.4, 126.1, 128.0,

129.2, 147.1. Anal. Calcd for C₂₂H₂₁N₃: C, 80.70; H, 6.46; N, 12.83.

Found: C, 79.96; H, 6.58; N, 11.78.

4,4,4-Triphenylbutan-1-amine (10). The title compound was

prepared using an adaptation of the procedure reported by

Dockendorff et al.⁴³Triphenylphosphine (603 mg, 2.3 mmol) was

added to a solution of 1,1′,1″-(4-azidobutane-1,1,1-triyl)tribenzene 60

(150 mg, 0.46 mmol) in THF/H₂O (5:0.5 mL). The reaction mixture

was stirred at 60 °C for 12 h and then concentrated in vacuo. The

crude product was purified by flash chromatography [SiO₂, 3−15%

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cultured in RPMI (Invitrogen, Paisley, U.K.), supplemented with 10%

fetal bovine serum (PAA, Pasching, Austria). BxPC-3 (ATCC CRL-

1687) cells were cultured in RPMI (Invitrogen, Paisley, U.K.),

supplemented with 1% nonessential amino acids (Invitrogen, Paisley,

U.K.), 1% sodium pyruvate (Invitrogen, Paisley, U.K.), 1% glutamine

(Invitrogen, Paisley, U.K.), and 10% fetal bovine serum (PAA,

Pasching, Austria). hTERT-HME1 cells (Clontech, Basingstoke, U.K.)

were cultured in mammary epithelial cell growth medium (PromoCell,

Heidelberg, Germany). All cells were maintained at 37 °C, 95%

humidity, and 5% carbon dioxide in a humidified incubator. They were

used for experiments for 6−8 weeks before they were replaced with

fresh stocks that were stored in liquid nitrogen.

Proliferation Assays. Cells were seeded in triplicate in 96-well

assay plates at 1.250 cells (BxPC-3, HCT116), 2.500 cells (hTERT-

HME1, NCI-H1299), or 5.000 cells (K562) per well in 100 μL of the

respective growth medium. Medium blanks and cell blanks for every

cell line were also prepared. On the next day, inhibitors were added

with a starting concentration of 100 μM in a 3-fold serial dilution

series. At 72 h after inhibitor addition, 10% Alamar blue (Invitrogen,

Paisley, U.K.) was added, and depending on the cell line, 2−12 h later

the absorbance was measured at 570 and 600 nm. All values were

corrected for the absorbance of the medium blank, and the corrected

cell blanks were set to 100%. Calculations for determining the relative

proliferation were performed using equations described in the

manufacturer’s manual. Finally, the GI₅₀values were determined

using a sigmoidal dose−response fitting (variable slope) with

GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego,

CA, U.S.).

Tumor Xenografts. The animal experiments were performed at

Oncotest GmbH with female NMRI nu/nu mice (Charles River,

Sulzfeld, Germany). Tumor fragments were obtained from xenografts

in serial passage in nude mice. After removal from donor mice, tumors

were cut into fragments (4−5 mm diameter) and placed in PBS until

subcutaneous implantation. Recipient mice were anesthetized by

inhalation of isoflurane. A small incision was made, and one tumor

fragment per animal was transplanted with tweezers. The approximate

age at implantation was 5−7 weeks. At 10−12 weeks, mice were

randomized to the various groups and dosing started when the

required number of mice carried a tumor of 50−250 mm³volume,

preferably 80−200 mm³. Vehicle details and drug formulation are

described in the Supporting Information.

Vehicle control mice (group 1) were treated with 10 mL/kg

rac-29 vehicle on days 0, 2, and 4 and with 7.5 mL/kg on days 17,

19, 24, 31, and 34. The rac-29 treatment group (group 2) received

20 mg/kg rac-29 on days 0, 2, and 4 and 15 mg/kg rac-29 on days 17,

19, 24, 31, and 34. All treatments were given intraperitoneally.

The experiment was terminated on day 34, and tumor samples were

collected.

of Eg5 with 25 appeared after 4 days in hanging drops by mixing 1 μL

of protein−inhibitor complex with 1 μL of reservoir solution

containing 22% polyethylene glycol-3350, 0.25 M ammonium sulfate,

0.1 M potassium sodium tartrate tetrahydrate, and 0.1 M MES, pH 5.8,

in VDX plates (Hampton Research) at 4 °C. A thick needle-shaped

crystal with dimensions of approximately 0.1 mm × 0.01 mm × 0.01

mm was immersed in cryoprotectant solution (27.6% polyethylene

glycol-3350, 0.36 M of ammonium sulfate, 0.12 M MES, pH 5.5,

0.06 M potassium chloride, and 20% erythritol) and flash frozen in

liquid nitrogen. Crystals of Eg5 with rac-29 appeared after 1 week in

hanging drops by mixing 1 μL of protein−inhibitor complex with

0.2 μL of 1 M potassium sodium tartrate tetrahydrate and 1 μL of

reservoir solution containing 23% polyethylene glycol-3350, 0.25 M

ammonium sulfate, and 0.1 M MES, pH 5.5, in VDX plates (Hampton

Research) at 4 °C. Dehydrating solution (33% polyethylene glycol-

3350, 0.25 M ammonium sulfate, 0.1 M potassium sodium tartrate

tetrahydrate, 0.1 M MES, pH 5.5, and 10% glycerol) was added slowly

to the crystal droplet until the total volume of the drop was 8 times the

original. The drop was then equilibrated against air in 4 °C for 30 min.

A cubic crystal with dimensions of approximately 0.1 mm on each side

was then flash frozen in liquid nitrogen.

Data Collection and Processing. Diffraction data for the Eg5−

inhibitor complexes Eg5−25 and Eg5−29 were recorded at the

European Synchrotron Radiation Facility (ESRF) beamlines ID23-1

and ID23-2, respectively. Data were processed using iMosflm⁴⁷and

scaled using Scala⁴⁸from the CCP4 suite of programs.⁴⁹The

structures were solved by molecular replacement (Phaser)⁵⁰using one

molecule of Eg5 motor domain from structures with PDB codes

1X88²⁰and 3KEN,¹⁹respectively. Refinement was carried out with

Phenix⁵¹and Refmac5, respectively. The calculation of R_freeused 5% of

data. Electron density and difference density maps, all σ_A-weighted,

were inspected, and the model was improved using Coot.⁵²Model

geometry was analyzed using Molprobity.⁵³For the Eg5−25 structure,

95.7% (2106) of the residues are in the preferred regions, 3.54% (78)

are in the allowed regions, and 0.77% (17) are outliers as shown by the

Ramachandran plot. For the Eg5−29 complex, 95.2% (316) of the

residues are in the preferred regions, 3.61% (12) are in the allowed

regions, and 1.20% (4) are outliers. Figures are prepared using

PyMOL.⁵⁴

ASSOCIATED CONTENT

■

S

* Supporting Information

Additional experimental procedures and compound character-

ization data, ADME profiling and tumor experiment methods,

supplementary results on acid stability of compounds, and

validation of Marfey’s method. This material is available free of

charge via the Internet at http://pubs.acs.org.

Mortality checks were conducted at least daily during routine

monitoring. Body weight was used as a means of determining toxicity.

Mice were weighed twice a week. The tumor volume was determined

by two-dimensional measurement with a caliper on the day of

randomization (day 0) and then twice weekly (i.e. on the same days on

which mice were weighed). Tumor volumes were calculated according

to the formula (ab²) × 0.5 where a represents the largest and b the

perpendicular tumor diameter.

Tumor inhibition for a particular day (T/C in %) was calculated

from the ratio of the median RTV values of test versus control groups

multiplied by 100%. For the evaluation of the statistical significance

of tumor inhibition, the Mann−Whitney U-test was performed.

Individual RTVs were compared on days on which the minimum

T/C was achieved, as long as sufficient animals were left for statistical

analysis or otherwise on days as indicated. By convention, p ≤ 0.05

indicates significance of tumor inhibition. Statistical calculations were

performed using GraphPad Prism 5.01 (GraphPad Software, San

Diego, CA, U.S.).

Accession Codes

^†Coordinate and structure factor files for the Eg5−25 and

Eg5−29 complexes (PDB codes 4A51 and 4A50) were

deposited at the PDB.

AUTHOR INFORMATION

■

Corresponding Author

*For F.W.: phone, +86(0)-10-64852570; e-mail, wangfang@

moon.ibp.ac.cn. For F.K.: phone, +44(0)-141-3303186; fax,

+44-141-9426521; e-mail, f.kozielski@beatson.gla.ac.uk.

Present Addresses

^⊥Institute of Biophysics, Chinese Academy of Sciences, 15

Datun Road, Chaoyang District, Beijing, 100101, China.

^#Institute of Molecular and Cell Biology, 61 Biopolis Drive,

Proteos, 138673, Singapore.

Crystallization of the Eg5−25 and Eg5−29 Complexes.

Eg5₁₋₃₆₈was cloned, expressed, and purified as previously described.¹⁴

Purified Eg5 (20 mg/mL), in complex with 1 mM Mg²⁺ATP, was

incubated with 2 mM 25 or rac-29 (in DMSO) for 2 h on ice. Crystals

Author Contributions

^∥These authors contributed equally.

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Journal of Medicinal Chemistry

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caspase-independent cell death in K562 human chronic myeloid

leukemia cell line. Cancer Lett. 2010, 298, 99−106.

ACKNOWLEDGMENTS

■

We are grateful to Ruangelie Edrada-Ebel at Strathclyde

University, U.K., for performing HR-MS experiments, Denise

Gilmour for elemental analysis, Craig Irving for NMR support,

and Marta Kozlowska and Sandeep Talapatra from The

Beatson Institute for Cancer Research for providing purified

human kinesins to analyse the specificity of rac-29. We thank

Dr. Alexander Popov and Dr. Gordon Leonard of ESRF and of

EMBL-Grenoble for assistance and support in using beamlines

ID23-1 and ID23-2. We are grateful to the NCI/NIH for

providing some of the STLC analogues investigated in this

work. We thank Cancer Research UK for financial support.

(10) Kozielski, F.; Skoufias, D. A.; Indorato, R. L.; Saoudi, Y.;

Jungblut, P. R.; Hustoft, H. K.; Strozynski, M.; Thiede, B. Proteome

analysis of apoptosis signaling by S-trityl-^L-cysteine, a potent reversible

inhibitor of human mitotic kinesin Eg5. Proteomics 2008, 8, 289−300.

(11) Ding, S.; Nishizawa, K.; Kobayashi, T.; Oishi, S.; Lv, J.; Fujii, N.;

Ogawa, O.; Nishiyama, H. A potent chemotherapeutic strategy for

bladder cancer: (S)-methoxy-trityl-^L-cystein, a novel Eg5 inhibitor.

J. Urol. 2010, 184, 1175−1181.

(12) Xing, N.-D.; Ding, S.-T.; Saito, R.; Nishizawa, K.; Kobayashi, T.;

Inoue, T.; Oishi, S.; Fujii, N.; Lv, J.-J.; Ogawa, O.; Nishiyama, H. A

potent chemotherapeutic strategy in prostate cancer: S-(methoxy-

trityl)-^L-cysteine, a novel Eg5 inhibitor. Asian J. Androl. 2011, 13, 236−

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ABBREVIATIONS USED

■

CD, circular dichroism; DMF, dimethylformamide; DMSO,

dimethylsulfoxide; EC₅₀, half-maximal effective concentration;

FDVA, N_α-(2,4-dinitro-5-fluorophenyl)-L-valinamide; FMOC,

fluorenylmethyloxycarbony; GI₅₀, the concentration required to

achieve 50% growth inhibition; h, hour; hERG, human ether-

ago-go-related gene; HPLC, high performance liquid chroma-

tography; IC₅₀, median inhibitory concentration; K_i^app, apparent

K_i; KSP, kinesin spindle protein; MT, microtubule; SAR,

structure−activity relationship; STDC, S-trityl-^D-cysteine;

STLC, S-trityl-^L-cysteine; MDR, multidrug resistance; NCI,

National Cancer Institute; nd, not determined; ni, no

inhibition; PAMPA, parallel artificial membrane permeability

assay; PBS, phosphate buffered saline; Pgp, P-glycoprotein; rt,

room temperature; T/C, relative test tumor versus control

value; TFA, trifluoroacetic acid; THF, tetrahydrofuran

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