Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2] thiazolo[5,4- d ]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent

Article abstract of DOI:10.1021/jm200629m

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)- N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl) -2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

Full text of DOI:10.1021/jm200629m

ARTICLE
pubs.acs.org/jmc
Discovery of (+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-
oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-
methylbenzamide (AZD4877), a Kinesin Spindle Protein Inhibitor and
Potential Anticancer Agent
Maria-Elena Theoclitou,*,† Brian Aquila,‡ Michael H. Block,‡ Patrick J. Brassil,‡ Lillian Castriotta,‡
Erin Code,‡ Michael P. Collins,‡ Audrey M. Davies,‡ Tracy Deegan,‡ Jayachandran Ezhuthachan,‡,§
||
Sandra Filla,‡, ,§ Ellen Freed,‡,§,^ Haiqing Hu,‡ Dennis Huszar,‡ Muthusamy Jayaraman,# Deborah Lawson,‡
Paula M Lewis,‡ Murali V. P. Nadella,‡ Vibha Oza,‡ Maniyan Padmanilayam,# Timothy Pontz,‡
Lucienne Ronco,‡,§ Daniel Russell,‡ David Whitston,‡ and Xiaolan Zheng‡
†Cancer & Infection Research Area, AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom
‡Cancer & Infection Research Area, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
S Supporting Information
b
ABSTRACT: Structureꢀactivity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-
3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877),
from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical
potency and pharmaceutical properties suitable for clinical development. The selected compound
arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of
KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo
efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.
irst generation antimitotic drugs, such as taxanes and vinca
alkaloids, have demonstrated single-agent activity and also
proteins. The expression profile of KSP is indicative of its role in
mitosis. KSP is expressed in the thymus, tonsils, testes and bone
marrow and is absent from human central nervous system
neurons, which are postmitotic.11 Negligible levels of KSP are
detectable in adult nonproliferating tissue, while expression is
prominent in proliferating tissue during development.14ꢀ16 The
only known postdevelopment role for KSP is in the prophase of
mitosis, where it is required for centrosome separation and the
formation of a bipolar mitotic spindle.10,17 Inhibition of KSP in
proliferating tumor cells results in inhibition of centrosome
separation and consequent formation of a monopolar, or mono-
astral, spindle,10,18 arresting cells in mitosis and ultimately
leading to cell death. Since KSP inhibition has no apparent effect
on microtubules in nondividing cells, KSP inhibitors are not
expected to cause peripheral neuropathy, a side effect attributed
to disruption of the neuronal tubulin network.19 KSP is also not
expressed in the adult central/peripheral nervous system,11
consequently limiting the central nervous system side effect
profile of its inhibitors.
F
form the basis for a number of established combination therapies
in cancer therapy.1 Agents of this type exhibit a broad range of
clinical effectiveness by disrupting spindle dynamics, leading to
mitotic arrest and apoptosis.2,3 While effective, these drugs also
have significant toxicological limitations. Microtubules are vital in
nonproliferating cells, required for such processes as cell signaling
and vesicular transport, thus tubulin-targeting agents often demon-
strate toxicities such as neurotoxicity and peripheral neuropathy.4,5
Furthermore, innate and acquired resistance is a significant limita-
tion of current therapies due to tumor cells becoming resistant to
microtubule poisons through a variety of mechanisms, inclusive of
tubulin mutations.6ꢀ8 A clinical opportunity hence exists for novel
antimitotic approaches that are more specific and have the potential
to overcome observed toxicities and mechanisms of resistance
commonly seen with microtubule targeting drugs.
Mitotic kinesins have gained significant attention as new
targets for cancer therapy. Mitotic kinesins are molecular
motor proteins which are essential for the assembly and function
of the mitotic spindle.9ꢀ12 623 kinesins have been identified to
date, and these have been categorized into 14 different fam-
ilies. Kinesin spindle protein (KSP) or HsEg5 (Homo sapiens
Eg5)10,13,14 is a member of the bimC subfamily of kinesin related
In addition, overexpression of KSP levels has been observed in
a variety of human solid tumors, inclusive of breast, lung, ovarian,
Received: May 18, 2011
Published: September 07, 2011
r
2011 American Chemical Society
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ARTICLE
Figure 1. Structure of monastrol.
Figure 3. Modifications to five and six membered scaffolds examined
(R1ꢀR5).
synthesis could be used to obtain the large diversity of fused core
scaffolds necessary and hence each intermediate synthetic route
is outlined separately.
The main preparation of compounds 2ꢀ53, containing the
3H-thieno[2,3-e]pyrimidin-4-one core, is outlined in Scheme 1.
Methyl 3-aminothiophene-2-carboxylate was acylated to gener-
ate methyl 3-(butanoylamino)thiophene-2-carboxylate (step a),
which subsequently underwent cyclization (step b), followed by
alkylation (step c) with the respective R2 diverse reagents to yield
scaffold A. Subsequent bromination of these compounds (step d)
gave yield to the intermediates desired. These compounds were
then converted into the desired final compounds (compounds
2ꢀ53) via bromide displacement (with the desired R4 diverse
amines (step e)) and acylation (step f), to ultimately include the
respective R5 moiety. Final products were obtained via deprotec-
tion, if necessary.
Figure 2. Structure of compound 1 (AZD4877).
colon and bladder cancers, and leukemias.20ꢀ22 However, this
has often been observed in the context of a high mitotic index and
corresponding increases in expression of other mitotic markers
and may be primarily a reflection of a higher proliferative rate.20,21
All of these observations render KSP a very attractive phar-
macological target with the potential for inhibitors to exhibit
broad spectrum antitumor efficacy, matched with improved
tolerability, alongside activity in drug-resistant patient popula-
tions, compared to current microtubule focused agents.
Interest in mitotic kinesins as oncological drug targets was
instigated by the initial identification of monastrol (Figure 1) as a
selective KSP inhibitor.18 Since its discovery, a variety of potent
KSP inhibitors have been reported that inhibit via an allosteric
mode of inhibition (including SB-715992, SB-743921, CK010-
6023, MK-0731, ARRY-520 and HR22C16-A1;23ꢀ37 the data on
the large majority of these inhibitors have been published after
the period during which the current work described within this
paper was conducted). These inhibitors block enzymatic and
cellular functions resulting in decreased cellular proliferation,
mitotic arrest and cell death.28 SB-715992, CK0106023, MK-0731
and HR22C16-A1 have all been shown to possess antitumor
activity in xenograft models of cancer.30,31,35ꢀ40 KSP inhibitors
remain effective in tumor cells resistant to paclitaxel, as recent
results have also demonstrated.28,39
Intermediate compounds of the nature of scaffold B (which
ultimately would lead to generation of compounds 2ꢀ53) could
also be obtained via a parallel route (Scheme 1). This route namely
involved coupling of methyl 3-aminothiophene-2-carboxylate (same
starting material used to generate scaffold A) with 2-[(2-methyl-
propan-2-yl)oxycarbonylamino]butanoic acid (step h), followed
by cyclization (step i) and alkylation with the desired R2 diverse
reagents (step j). Final deprotection (step k) and reductive amina-
tion (step l) with the respective aldehydes (incorporating R4
diversity) resulted in scaffold B, which could be converted, as
previously, to the final desired compounds via acylation (incor-
porating R5 diversity (step f)) and deprotection (step g, if required).
Schemes 2 and 3 outline the synthetic procedures used to
generate compounds 54ꢀ61, 63, 64 (containing variations to the
heterocyclic core). The furan scaffold analogue was generated via
Scheme 2 (steps 1aꢀ1j). Furan-3-carboxylic acid was reacted
under Curtius conditions to generate N-furan-3-ylbutanamide
(steps 1a, 1b). VilsmeierꢀHaack reaction (step 1c) followed by
conversion of the resultant aldehyde into the cyano analogue
(step 1d), cyclization (step 1e) and subsequent benzylation
(R2 = (Ph)) (step 1f) yielded scaffold C. Conversion into the
desired final compound was accomplished in a similar manner to
the analogue outlined in Scheme 1, namely, bromination, intro-
duction of R4 via amine displacement (R4 = (ꢀ(CH2)3NHBoc)),
acylation to introduce R5 (R5 = (4-Me Ph)) and final protecting
group removal to yield compound 54 (steps 1gꢀ1j).
Herein we describe the discovery of a class of potent, selective
inhibitors of KSP, from which was derived our ultimate compound.
Compound 1 (AZD4877)41,42 (Figure 2) exhibits favorable phar-
macokinetic parameters and activity in a pharmacodynamic
model consistent with a potential therapeutic role in the treat-
ment of neoplastic disease.
The pyrrole analogue scaffold was produced via Scheme 2
(steps 2aꢀ2k). Isoxazole was reacted with diethyl 2-aminopro-
panedioate to generate ethyl 3-amino-1H-pyrrole-2-carboxylate
(step 2a). Acylation, cyclization and subsequent benzylation
(R2 = (Ph)) offered scaffold D (steps 2bꢀ2e). Application of
the same procedure as Scheme 2 (step 1g) led to bromination of
the five membered heterocyclic ring only. Utilization of a
different bromination procedure (Scheme 2, step 2g) allowed
the introduction of bromide in the desired position. Introduction
’ CHEMISTRY
From extensive analysis of the sparse KSP inhibition literature
information available at the time of project commencement,43
the initial aim of the chemistry effort was to synthesize KSP
inhibitors of the nature shown in Figure 3 in order to obtain
structureꢀactivity relationships at the various positions of diver-
sity around the central fused pyrimidinone core. No generic
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Scheme 1. Synthesis of Compounds Containing 3H-Thieno[2,3-e]pyrimidin-4-one Core (Compounds 2ꢀ53)a
a Conditions: (a) butanoyl chloride, CH2Cl2, Et3N, 0 ꢀC, 30 min (99%); (b) NH4OH, pressure reactor, 105 ꢀC, 3 h (70%); (c) R2CH2Br, K2CO3, DMF,
25 ꢀC, 12 h; (d) Br2, NaOAc, AcOH, 55 ꢀC, 30 min; (e) R4NH2, K2CO3, DMF, 55 ꢀC, 1 h; (f) R5COCl, CH2Cl3, Et3N, 25 ꢀC, 30 min; (g, if required)
TFA, CH2Cl2, 25 ꢀC, 30 min; (h) CH2Cl2, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-ethyl-N-propan-2-ylpropan-2-amine,
DMAP, rt, 48 h (74%); (i) 30% NH4OH, 95 ꢀC, 2 h (50%); (j) R2 CH2Cl, K2CO3, DMF, rt, 16 h; (k) HCl, dioxane, rt, 2 h; (l) tert-butyl N-(3-
oxopropyl)carbamate, C2H4Cl2, Et3N, rt, 30 min then sodium triacetoxyborohydride rt, 1 h.
of R4 was achieved via amine displacement (R4 = (ꢀ(CH2)3-
NHBoc)) (step 2h), and acylation introduced R5 (R5 = (4-Me Ph))
(step 2i). Dehalogenation of the heterocyclic ring was carried out
prior to final protecting group removal to generate compound 55
(steps 2j, 2k).
quinazolinone analogue (scaffold F (R2 = (Ph))). The 3-amino-
pyrazine-2-carboxylic acid analogue compound (W = N) was
generated in an identical fashion (scaffold G (R2 = (Ph))). The
terminal desired compounds were produced via similar reactions
to those mentioned previously (steps 4dꢀ4g), incorporating R4
(R4 = (ꢀ(CH2)3NHBoc)) and R5 (R5 = (4-Me Ph)), to produce
60 (W = C) and 61 (W = N) respectively.
The isoxazole scaffold analogue (scaffold H) was generated via
synthesis of 5-(butanoylamino)-3-methyl-1,2-oxazole-4-carbox-
amide from commercially available starting materials (Scheme 2
(step 5a)). Cyclization and benzylation (R2 = (Ph)) gave yield to
scaffold H (Scheme 2 (steps 5b, 5c)), which was ultimately
converted to the final desired product 63 via similar steps
to those mentioned previously (R4 = (ꢀ(CH2)3NHBoc), R5 =
(4-Me Ph)) (steps 5dꢀ5g).
Synthesis of substituted analogues of 3H-thieno[3,2-e]pyri-
midin-4-one was accomplished in a similar manner, and the main
scaffold synthesis is shown in Scheme 2 (steps 3aꢀ3d). Starting
materials 1,4-dithiane-2,5-diol and 2-cyanoacetamide were utilized
to generate 2-aminothiophene-3-carboxamide (step 3a), which
was then acylated, cyclized and benzylated (R2 = (Ph)) to give
the intermediate scaffold E (steps 3bꢀ3d). The synthetic routes
for compounds 56ꢀ59 are shown fully in Scheme 3. Bromina-
tion of scaffold E led to a dibrominated intermediate (Scheme 3,
steps a, b). This compound was progressed via amine displace-
ment (R4 introduction; R4 = (ꢀ(CH2)3NHBoc)) and acylation
(R5 introduction, R5 = (4-Me Ph)) (steps c, d) and subsequently
generated compounds 56 (via deprotection) (step e) and 57 (via
dehalogenation followed by deprotection) (steps f, g). Chiral
purification of 56 generated 66 and 67. Scaffold E also served to
generate 58 and 59. Chlorination of scaffold E followed by
bromination led to a mixture of compounds shown in Scheme 3
(steps h, i). Progression of this mixture via amine displacement
(R4 introduction; R4 = (ꢀ(CH2)3NHBoc)), acylation (R5 intro-
duction, R5 = (4-Me Ph)) and deprotection ultimately gave yield
to 58 and 59 (steps jꢀl).
Synthesis of N-(3-aminopropyl)-N-[1-(6-benzyl-3-methyl-7-
oxo-[1,2]thiazolo[4,5-d]pyrimidin-5-yl)propyl]-4-methylbenza-
mide (64) (Scheme2 (steps 6aꢀ6m)) was lengthy. Ethyl 3-amino-
2-methanethioylbut-2-enoate was used togenerateethyl 3-methyl-
1,2-thiazole-4-carboxylate (steps 6a, 6b), which was subsequently
converted to 3-methyl-1,2-thiazole-4-carboxylic acid (steps 6c,
6d). Employment of the Curtius reaction led to synthesis of tert-
butyl N-(3-methyl-1,2-thiazol-4-yl)carbamate (step 6e), which
was then converted to 3-methyl-4-[(2-methylpropan-2-yl)oxy-
carbonylamino]-1,2-thiazole-5-carboxylic acid (step 6f). This
compound was deprotected and converted to 3-methyl-5-
propyl-[1,2]thiazolo[4,5-d][1,3]oxazin-7-one (steps 6g, 6h), by
the use of butanoyl chloride. Reaction with benzylamine (step 6i)
gave yield to scaffold I (R2 = (Ph)) and subsequent steps as
The six membered ring analogues were synthesized via cyclic
oxazinones. Namely, in Scheme 2 (steps 4aꢀ4c), 2-aminopyr-
idine-3-carboxylic acid (W = C) was acylated and converted
into the oxazinone, before being converted into the desired
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ARTICLE
Scheme 2. Synthesis of Compounds Containing Variations to the Heterocyclic Corea
a Conditions: (1a) toluene, Et3N, diphenylphosphoryl azide, 5ꢀ10 ꢀC, 30 min, reflux 3 h; (1b) propyl magnesium chloride, 0ꢀ5 ꢀC, 1 h (two steps
35%); (1c) POCl3, DMF, 0ꢀ5 ꢀC, 1.5 h (68%); (1d) NH2OH, 4-methylbenzenesulfonic acid, MgSO4, 55 ꢀC, 16 h (77%); (1e) KOH, t-BuOH, reflux,
3 h (60%); (1f) bromomethylbenzene, K2CO3, tetrabutylammonium hydrogen sulfate, toluene, rt, 3 h, (61%); (1g) Br2, NaOAc, AcOH, 50 ꢀC, 30 min
(88%); (1h) tert-butyl N-(3-aminopropyl)carbamate, Et3N, CH2Cl2, 25 ꢀC, 5 days (99%); (1i) 4-methylbenzoyl chloride, Et3N, CH2Cl2, 25 ꢀC, 30 min
(93%); (1j) TFA, CH2Cl2, 25 ꢀC, 30 min (67%); (2a) diethyl aminomalonate hydrochloride, EtOH, NaOEt, 8 ꢀC, 30 min, then acetic acid, NaOAc, 25 ꢀC,
48 h (38%); (2b) butanoyl chloride, CHCl3, pyridine, 0 ꢀC, 1 h (97%); (2c) benzyltributylammonium bromide, (CH3)2SO4, CH2Cl2, 50% NaOH,
0ꢀ25 ꢀC, 1 h (84%); (2d) 30% NH4OH, 110ꢀ115 ꢀC, 6 h (19%); (2e) K2CO3, toluene, tetrabutylammonium hydrogen sulfate, reflux, 16 h (both N-
and O-benzylated products obtained); (2f) Br2, NaOAc, AcOH, 65 ꢀC, 5 min (96%); (2g) CCl4, N-bromosuccinimide, 2,20-azobisisobutyronitrile,
reflux, 45 min (95%); (2h) tert-butyl N-(3-aminopropyl)carbamate, Et3N, CH2Cl2, 25 ꢀC, 5 days (36%); (2i) 4-methylbenzoyl chloride, Et3N, CH2Cl2,
25 ꢀC, 12 h (90%); (2j) 5%Pd/C, H2, EtOH, 25 ꢀC, 18 h (53%); (2k) TFA, CH2Cl2, 25 ꢀC, 30 min (86%); (3a) 2-cyanoacetamide, EtOH, Et3N, 50 ꢀC,
3 h (72%); (3b) butanoyl chloride, CH2Cl2, Et3N, 25 ꢀC, 12 h (96%); (3c) 2 N NaOH, reflux, 1 h (88%); (3d) bromomethylbenzene, K2CO3, DMF,
25 ꢀC, 12 h (62%); (4a) butanoyl butanoate, reflux, 1 h (W = C 64%, W = N 59%); (4b) 30% NH4OH, 100 ꢀC, 18 h (W = C 80%, W = N 52%); (4c)
bromomethylbenzene, K2CO3, DMF, 25 ꢀC, 12 h (both N- and O-benzylated products obtained); (5a) butanoyl butanoate, 150 ꢀC, 0.5ꢀ1 h (87%);
(4d) Br2, NaOAc, AcOH, 45 ꢀC, 30 min; (4e) tert-butyl N-(3-aminopropyl)carbamate, K2CO3, DMF, 60 ꢀC, 4 h; (4f) 4-methylbenzoyl chloride,
CH2Cl2, Et3N, 25 ꢀC; (4h) TFA, CH2Cl2, 25 ꢀC, 30 min; (5b) 2 N aq NaOH, microwave, 140 ꢀC, 20 min (74%); (5c) bromomethylbenzene, K2CO3,
DMF, 25 ꢀC, 16 h (68%); (5d) Br2, NaOAc, AcOH, 100 ꢀC, 48 h (62%); (5e) tert-butyl N-(3-aminopropyl)carbamate, K2CO3, CH3CN, 100 ꢀC, 16 h
(74%); (5f) 4-methylbenzoyl chloride, CH2Cl2, Et3N, 25 ꢀC, 1 h (76%); (5g) HCl, dioxane, 25 ꢀC, 2 h (93%); (6a) POCl3, DMF, THF, 0 ꢀC, 30 min;
(6b) NaSH, CH2Cl2, rt (steps 6a and 6b, 74%); (6c) 3-chlorobenzenecarboperoxoic acid, EtOH, 75 ꢀC, 2 h (93%); (6d) aq NaOH, THF, rt, 16 h (79%);
(6e) t-BuOH, Et3N, diphenylphosphoryl azide, reflux, 16 h (97%); (6f) LDA/THF, ꢀ78 ꢀC, 4 h, then CO2, rt, 16 h (39%); (6g) HCl, dioxane, rt, 16 h
(100%) (6 h) butanoyl chloride, pyridine, 0 ꢀC to rt, 16 h, then 2 M HCl (64%); (6i) phenylmethanamine, microwave, 200 ꢀC, 20 min (71%); (6j) Br2,
NaOAc, AcOH, 100 ꢀC, 30 min (99%); (6k) tert-butyl N-(3-aminopropyl)carbamate, N-ethyl-N-propan-2-ylpropan-2-amine, DMF, rt, 1 h; (6l)
4-methylbenzoyl chloride, CHCl3, N-ethyl-N-propan-2-ylpropan-2-amine, 60 ꢀC, 12 h; (6m) HCl, dioxane, rt, 30 min (steps k, l, m 16%).
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Scheme 3. Synthesis of Compounds 56ꢀ59a
a Conditions: (a) Br2, NaOAc, AcOH, 45 ꢀC, 30 min (60%); (b) Br2, NaOAc, AcOH, 55 ꢀC, 19.5 h (81%); (c) tert-butyl N-(3-
aminopropyl)carbamate, K2CO3, DMF, 60 ꢀC, 4 h (53%); (d) 4-methylbenzoyl chloride, CH2Cl2, Et3N, 25 ꢀC, 3 h (93%); (e) TFA, CH2Cl2,
25 ꢀC, 30 min (96%); (f) 5% Pd/C, H2, MeOH, 25 ꢀC, 2.5 h (28%); (g) TFA, CH2Cl2, 25 ꢀC, 30 min (98%); (h) N-chlorosuccinimide, DMF, 50
ꢀC, 3 days, then ice, 1 h (84%); (i) Br2, NaOAc, AcOH, 55 ꢀC, 19.5 h (progressed as mixture); (j) tert-butyl N-(3-aminopropyl)carbamate, K2CO3,
DMF, 60 ꢀC, 4 h (mixture); (k) 4-methylbenzoyl chloride, CH2Cl2, Et3N, 25 ꢀC, 3 h (mixture); (l) TFA, CH2Cl2, 25 ꢀC, 30 min (steps iꢀl:
compound 58, 38%; compound 59, 23%).
outlined previously (steps 6jꢀ6m) led to 64 (R4 = (ꢀ(CH2)3-
the use of the same intermediate utilized to synthesize 62,
namely, 5-amino-3-methyl-1,2-thiazole-4-carbonitrile. After
acylation with the appropriate reagent (step a, R3 = (Me)),
this compound was converted to scaffolds M, N and O
(Scheme 5; R2 = (Ph), (4-F Ph) and (3-F Ph) respectively)
via cyclization and benzylation with the appropriate reagent
(steps bꢀd). Displacement of the bromide with the respective
R4-substituted amine, as had been utilized previously for all
compounds (Schemes 2ꢀ4), could not be exploited in this
case as this procedure led to elimination. Another route was
hence devised via the conversion of the intermediate bro-
mide to the azide analogue (steps e, f). Staudinger reduction
(step g) gave yield to the desired amine analogue, and sub-
sequent reductive amination (step h; R4 = (ꢀ(CH2)3NH-
Boc)) resulted in the intermediate Boc-protected amine. Once
again, acylation with a variety of desired acyl chlorides (step i)
gave yield to 65 (following deprotection, step j) and 1, 70, 72, 74,
76, 78 (following chiral purification and subsequent deprotec-
tion, steps k, l).
NHBoc), R5 = (4-Me Ph)).
Scaffolds J, K and L (Scheme 4; R2 =(Ph), (4-FPh) and(3-FPh)
respectively) were synthesized from 5-amino-3-methyl-1,2-thiazole-
4-carbonitrile via consecutive acylation (step e), conversion of the
cyano moiety to an amide (step f), cyclization (step g) and finally
benzylation (step h; R2 = (Ph), (4-F Ph) and (3-F Ph) respectively).
Synthesisofthisrespectiveintermediatearosefrom the cyclizationof
(2E)-3-amino-2-cyanobut-2-enethioamide, which had been syn-
thesized via its precursor (2E)-2-cyano-3-ethoxybut-2-enethio-
amide (steps c, d). 2-(1-Ethoxyethylidene)propanedinitrile, syn-
thesized from commercial starting materials, had been utilized to
generate this precursor (steps a, b). Scaffolds J, K and L were then
brominated (step i), and R4 was introduced via amine displace-
ment (step j; R4 = (ꢀ(CH2)3NHBoc)). Acylation with a variety
of desired acyl chlorides (step k) gave yield to 62 (following
deprotection, step l) and 68, 69, 71, 73, 75, 77 (following chiral
purification and subsequent deprotection, steps m, n).
The synthetic route utilized to generate 1 and its respec-
tive analogues is outlined in Scheme 5. The route incorporated
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Scheme 4. Synthesis of Compounds 62, 68, 69, 71, 73, 75, 77a
a Conditions: (a) 1,1,1-triethoxyethane, glacial acetic acid, 85ꢀ140 ꢀC, 40 min (91%); (b) H2S, anhydrous benzene, Et3N, rt 40 min (25%); (c) 30%
NH3/MeOH, 25 ꢀC, 16 h (63%); (d) 30% hydrogen peroxide, MeOH, 60 ꢀC, 4 h (80%); (e) butanoyl chloride, CH2Cl2, 25 ꢀC, 16 h (95%); (f) 30%
NH3/MeOH, 30% hydrogen peroxide, 60 ꢀC, 16 h (72%); (g) 30% NH3/MeOH, 140 ꢀC, 4 h (34%); (h) R2CH2Br, K2CO3, DMF, 25 ꢀC, 16 h (R2 = Ph;
32%); (i) Br2, NaOAc, AcOH, 100 ꢀC, 20 min (R2 = Ph; 100%); (j) tert-butyl N-(3-aminopropyl)carbamate, N-ethyl-N-propan-2-ylpropan-2-amine,
EtOH, reflux, 16 h (R2 = Ph; 17%); (k) R5COCl, CH2Cl2, Et3N, 25 ꢀC, 30 min (R2 = Ph, R5 = 4-Me Ph; 94%); (l) HCl, ether, 25 ꢀC, 20 h (R2 = Ph, R5 =
4-Me Ph; 87%); (m) chiral purification (98% total recovery if respective enantiomers); (n) HCl, dioxane, rt, 20 min (100%).
’ RESULTS AND DISCUSSION
activity. Ortho- substitutions were the most unfavorable (4),
followed by para- and meta- position substitutions (6ꢀ8 and
5 respectively). Heterocyclic analogues (10ꢀ12) were less
potent than their aryl counterparts presumably due to their
decrease in topical lipophilicity. For example, replacement of
the phenyl core with a pyridine core proved detrimental, regard-
less of the location of nitrogen (3 (nanomolar activity) com-
pared to 10ꢀ12 (micromolar activity)). One example where
the benzylic group was replaced with an alkyl moiety (9) also
reduced potency (>75-fold decrease). Only the plain benzylic
core (3) had accepted potency and resulted in appropriate
cellular activity for compound progression. This site of modi-
fication (R2) was hence left unaltered for further explor-
atory work.
The separate positions of diversity were systematically varied
around the central core to determine specific structureꢀactivity
relationships. Most potent compounds described showed clear
cell cycle effects consistent with inhibition of KSP (monoastral
phenotype observed).
Initially various alkylation substitutions were carried out on
the N-(3-aminopropyl)-4-methyl-N-[1-(4-oxo-3H-thieno[3,2-d]-
pyrimidin-2-yl)propyl]benzamide scaffold (Table 1). Complete
removal of the R2 moiety rendered tested compounds with
micromolar activity. The active site pocket appeared to be very
spatially restricted in this area, as any minor substitution gave a
decrease in activity (3 compared to 4ꢀ8); even the 2-fluoro-
substituted analogue (4) resulted in significant changes in
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Scheme 5. Synthesis of Compounds 1, 65, 70, 72, 74, 76, 78a
a Conditions: (a) 3-methylbutanoyl chloride, pyridine, 0 ꢀC to rt, 16 h (79%); (b) 30% NH3/MeOH, hydrogen peroxide, 60 ꢀC, 16 h (71%); (c) 30%
NH3/MeOH, 140 ꢀC, 5 h (38%); (d) R2CH2Br, K2CO3, DMF, rt, 16 h (R2 = Ph; 70%); (e) Br2, NaOAc, AcOH, 100 ꢀC, 50 min (R2 = Ph; 99%); (f)
NaN3, DMF, rt, 1 h (R2 = Ph; 94%); (g) 5% Pd/C, H2, MeOH, rt (used without further purification); (h) tert-butyl N-(3-oxopropyl)carbamate, 4 Å
molecular sieves, CH2Cl2, rt, 3 h, then AcOH, sodium triacetoxyborohydride rt, 16 h (used without further purification); (i) R5COCl, CHCl3, pyridine,
rt, 48 h (steps g, h and i (R2 = Ph, R5 = 4-Me Ph; 32%)); (j) HCl, dioxane, rt, 20 min (R2 = Ph, R5 = 4-Me Ph; 99%); (k) chiral purification (R2 = Ph, R5 =
4-Me Ph; 48%); (l) HCl, dioxane, rt, 20 min (R2 = Ph, R5 = 4-Me Ph; 100%).
Variation of the amine side chains rendered similar structureꢀ
activity relationships (Table 2) with complete removal of the
amine side chain abolishing activity (15). The best side chain
included a linear linker with a terminal primary amine, ideally
with a three carbon length spacer (3 compared to 16). Fusions to
yield secondary cyclic amine side chains (17ꢀ19) only served
to give significant decreases in activity (3 compared to 17ꢀ19
resulted in >30ꢀ140-fold decrease), presumably due to steric
interactions. Conversions of the terminal primary amine also
decreased activity. A terminal amide (20), sulfonamide (21) or
carbamate (22) moiety significantly reduced activity, yielding
micromolar compounds as a consequence. Hardly any amine
side chain substitutions led to an increase in activity, hence no
further significant R4 changes were made to this linkage.
Distinct structureꢀactivity relationships were obtained with
substitutions of the pendant amide moiety (R5, Table 3). This
pocket appeared to have a requirement for increased lipophilicity
as well as necessitating certain structural requirements. Alkyl
amide substitutions, whether acyclic (23, 24) or cyclic (25ꢀ27)
resulted in micromolar activity. The effect of variation in
lipophilicity was also seen with five membered heteroaromatic
rings (45ꢀ47) as well as heteroaryl groups, such as substituted
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Table 1. StructureꢀActivity Relationships Resulting from
Table 2. StructureꢀActivity Relationships Resulting from
Variation of R2 (R3, R4 and R5 Are as Shown)
Variation of R4 (R2, R3 and R5 Are as Shown)
activity was obtained when the para-position was substituted
with either methyl (3) or bulky halogen substituents (30, 32).
The central fused pyrimidinone core was also varied to gen-
erate the large diversity of fused core final compounds necessary
(Table 4). Compounds generated, with this variation (3, 54ꢀ64),
contained the best moieties in positions R2, R4 and R5 as identified
previously (R2 = Ph, R4 = ꢀ(CH2)3NH2, R5 = 4-Me Ph). With
regard to the respective fused heterocyclic scaffolds, in general, the
six membered heterocyclic ring analogues (60, 61) exhibited lower
potency than the five membered ring analogues (3, 54ꢀ59,
62ꢀ64). The lower potency was presumably due to the lower
overall lipophilicity of the molecules synthesized.
Within the five membered heterocyclic ring series, com-
pounds of the nature of 3, 54 and 55 (originating from scaffolds
A, C and D respectively) all appeared to have similar potency,
regardless of the five membered heterocyclic ring moiety present.
In the thiophene series synthesized, compounds originating from
scaffold E (56) were slightly more active than their regioisomeric
counterparts originating from scaffold A (3). This was reflective
more in their cellular potency rather than their enzyme potency.
Substitutions with halogens at both the 2- and 3-positions of the
compounds originating from scaffold E (57ꢀ59) decreased the
overall potency from their unsubstituted analogue (56). This
avenue was not investigated further. The most potent com-
pounds from the five membered mono- or diheteroatom core
variants (3, 54ꢀ64) were deemed to be 56, 62 and 63 (based on
pyridine (48), which decreased potency significantly (compared
to 3, compounds 45 and 47 resulted in >400-fold decrease whereas
46 and 48 resulted in g1000-fold decrease). Bicyclic substitutions
gave differing results (49ꢀ53). Bicyclics that served to reduce
lipophilicity (49, 51) led to drastic decreases in activity. Com-
pounds that retained lipophilicity (50) appeared to maintain activity
with small activity variations arising from steric interactions.
The widest range of potency variations was observed with the
monocyclic aryl moieties (Table 3, 28ꢀ44). The plain phenyl
group (28) reduced activity (>200-fold decrease compared to 3),
also presumably due to the decrease in lipophilicity. Common
substitutions on the phenyl moiety rendered varying results.
Substitution around the ring proved to be most detrimental in
the ortho-position (29, 37) (>600-fold decrease compared to 3).
Meta-substitution also served to decrease activity (compared to
3, compound 34 resulted in <10-fold decrease whereas 36 resulted
in g300-fold decrease). Electron withdrawing and electron donat-
ing groups in the para-position did affect the potency, however
the activity observed appeared to be more correlated with the
lipophilicity of the respective moieties (30, 32, 39). The posi-
tioning of a large lipophilic group in the para-position gave rise to
the most potent compounds observed (3, 30, 32), presumably
through the access of the distant section of this lipophilic pocket.
Steric restrictions in this pocket led to a decrease in potency if
larger lipophilic groups were employed (isopropyl, 40). The best
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Table 3. StructureꢀActivity Relationships Resulting from Variation of R5 (R2, R3 and R4 Are as Shown)
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Table 4. StructureꢀActivity Relationships Resulting from Variation of the Fused Heterocyclic Core
Table 5. Solubility of Most Potent Compounds
further progress only compounds 56 and 62 as these compounds
exhibited millimolar solubility (compared to micromolar for 63)
and were thus better suited for the overall drug profile required.
The sole position of diversity that remained to be explored was
R3 (Figure 2). The variation of this moiety was intentionally left
to the latter stages for ease of synthesis. These compounds
required an entirely different synthetic route to be devised
(Scheme 5). R3 variation (R3 = (H) or (Me)) was thus restricted
to the isothiazole scaffold, as this was indicated as the most
promising core variation (from Table 5). Results (shown in
Table 6) indicated that the newly synthesized compound 65 (R3 =
(Me)) had very similar cellular potency to compound 62 (R3 =
(H)) and hence a distinction would have to be made between 56,
62 and 65 using further parameters to enable their progression.
From the overall data available (Tables 1ꢀ6), 56, 62 and 65
stood out as the most potent and promising compounds for
compd
solubility (μM)
56
62
63
1239
1572
69
a combination of their enzyme and cellular potency) and hence
were progressed further to determine their respective physical
properties and pharmacokinetic parameters, ultimately contri-
buting to their in vivo efficacy.
The program goal was an intravenous (iv) agent, and hence
aqueous solubility was vitally important. The solubility of the
three initial leads chosen (56, 62, 63) was thus measured
(Table 5). From the results outlined, the decision was made to
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progression to in vivo models. Due to the fact that these com-
pounds were chiral in nature, they were subsequently separated
into their respective enantiomers (1, 66ꢀ70), and their enzyme
and cellular activities are shown in Table 7. The most active
enantiomer (1, 66, 68) was always the (+) antipode (when
dissolved at a concentration of 1 mg/mL in methanol, at 20.0 ꢀC
measured at 589 nm). Based on the cellular potencies of the
respective enantiomers, 62 and 65 were chosen for progression.
In order to further explore the isothiazole scaffold exhibited in
compounds 62 and 65, a distinct set of isothiazole compounds
was synthesized, in parallel, encompassing the best variants
identified at each R group position (Tables 1, 2, 3 and 6; R2 =
(Ph), (4-F Ph), (3-F Ph); R3 = (H), (Me); R4 = (ꢀ(CH2)3-
NH2); R5 = (4-Me Ph), (4-Br Ph), (3-F, 4-Me Ph)). All of these
analogues were synthesized by incorporation of the desired
R2ꢀR5 groups into the respective steps outlined in Schemes 4
and 5. The final enantiomers were isolated for comparison
(71ꢀ78), and their cellular potencies are shown in Table 8.
In order to determine the ultimate compounds for progres-
sion, combined enzyme and cellular data (outlined in Tables 7
and 8) were utilized, and a decision was made to further progress
compounds 1 and 68 as the best candidates.
The lower in vivo clearance (nude mouse) of 1 in comparison
to 68 (17 mL/min/kg vs 50 mL/min/kg; Table 9) served to aid
the project in determining 1 as the ultimate compound to
eventually progress into our pharmacodynamic model.
Prior to its assessment in our pharmacodynamic model to
determine its in vivo efficacy, further analysis of its pharmacoki-
netic properties was carried out.
Cytochrome P450 (CYP450) Inhibition. The potential for 1
to inhibit human cytochrome P450s was assessed in vitro using
specific substrates, recombinant enzymes and analysis by mass
spectrometry. For all five human isoforms tested (CYP1A2, 2C9,
2C19, 2D6 and 3A4), IC50 values obtained were >10 μM. The
potential for a clinically significant effect of 1 on the disposition
of other concomitant medications was hence considered low.
Kinesin and Kinase Selectivity. We were seeking to identify a
small molecule inhibitor of KSP, which was selective in relation
to other kinesins. To avoid nonspecific activity against other
targets, the compound also had to be selective versus a diverse
range of enzymes, receptors, ion channels and transporters,
inclusive of kinases. The activity of 1 was hence evaluated against
human recombinant KSP and two other selected kinesins in
vitro. Mitotic kinesin-like protein (MKLP) and conventional
kinesin were significantly less sensitive to 1, exhibiting IC50
values of 50 μM and IC50 of >100 μM respectively.
Table 6. StructureꢀActivity Relationships Resulting from
Variation of R3
In a separate panel, 1 (at a 10 μM concentration) did not sig-
nificantly inhibit any of 25 kinases screened (confidential data). The
highest inhibition was seen with lymphocyte specific protein tyrosine
kinase (LCK) and mitogen-activated protein kinase-activated pro-
tein kinase 1a (MAPKAP-K1a) at 42% and 35% respectively.
1 was also tested in a panel of 62 in vitro radioligand binding
and enzyme assays covering a diverse range of enzymes, recep-
tors, ion channels and transporters at a single concentration of
10 μM (company: MDS Pharma Services, Lead Profiling Screen,
available in the Supporting Information). Significant activity
(defined as >50% inhibition) was detected in only 5 of the 62
in vitro radioligand binding and enzyme assays (namely, 5-lipoxy-
genase, somatostatin receptor 1, two sites of the L-type calcium
compd
R3
cell IC50 (μM)
62
65
H
0.004
0.002
Me
Table 7. Enzyme and Cellular Potency of Respective Enantiomers (Compounds 1, 66ꢀ70)
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Table 10. Effects of Compound 1 in in Vitro Enzyme and
Table 8. Distinct Set of Isothiazole Compounds, Encom-
Radioligand Binding Assaysa
passing the Best Variants at Each R2ꢀR5 Group Position
% inhibition
at 10 μM of
target
sodium channel, site 2*
compd 1
99
87
80
76
71
L-type calcium channel, benzothiazepine site*
somatostatin sst1 receptor
L-type calcium channel, phenylalkylamine site*
5-lipoxygenase
a All human except where noted with an asterisk (*).
Table 11. Plasma Protein Binding of Compound 1
plasma
plasma protein binding (% bound)
rat (Wistar)
human
95.6
95.4
clearance. Intrinsic clearance (CLint) values, predicted CL values
and in vivo CL values are presented in Table 12.
In general, both microsomes and hepatocytes provided a
reasonable prediction of total in vivo clearance for preclinical
species tested.
The pharmacokinetic profile of 1 was assessed in rat following iv
bolus dosing at 1 and 5 mg/kg. Clearance values were moderate in
rat (50% of hepatic blood flow), and the volume of distribution
was high (10 L/kg). The observed t1/2 (half-life) was 3.5 h.
The pharmacokinetic properties of 1 following a single iv bolus
administration in rat are shown in Table 13.
In both male and female rats, the exposure of 1 was found
to increase with increasing dose between 1 and 6 mg/kg. No
differences in exposure were observed between day 1 and day 5
in a 5-day repeat dose study with single daily doses of 1 mg/kg.
Pharmacodynamic Activity in a Rat Hollow Fiber Model.
Hollow fibers seeded with Colo205 cells were implanted sub-
cutaneously into rats to assess the pharmacodynamic activity of 1.
Rats were treated with a single intravenous dose of the compound
(6 mg/kg and 12 mg/kg) or vehicle. A statistically significant
reduction in cell viability (Figures 4A and 5AꢀD) and increase in
cleaved caspase 3 levels (data not shown) was seen by 48 h
postdose at both dose levels. Increased numbers of monoasters
were observed at both time points after treatment with 1(Figures 4B
and 5E,F), and the number of phospho-histone-H3 (PHH3)
positive cells was significantly increased at 24 h (Figures 4C and
5G,H). These observations are consistent with KSP inhibition
and mitotic block resulting in cell death. Plasma levels of 1 were
higher than that detected in fibers 2 h after dosing, but drug levels
remained relatively constant in the fibers over 24 h resulting in
higher levels than that detected in plasma (Figure 6). At the
6 mg/kg dose level this corresponded to a total average drug fiber
concentration of 30.7 ng/mL (61 nM) and plasma concentration
of 2.8 ng/mL (5.6 nM) 24 h postdose.
Table 9. In Vivo Clearance of Compounds 1 and 68
nude mouse clearance
(mL/min/kg)
nude mouse
compd
compd
clearance (mL/min/kg)
1
17
68
50
channel and also the sodium channel, Table 10). In follow-
up studies, performed to explore the concentrationꢀresponse
relationship for 1 at these targets, the IC50 for inhibition of
the calcium channel activity (target: L-type calcium channel,
guinea-pig ileum) was 2.7 μM and for the sodium channel
(target: sodium channel, guinea-pig atria) was >30 μM. These
values were several hundred fold above the enzyme IC50 and
hence were not likely to be pharmacologically meaningful.
Pharmacokinetics. Plasma protein binding was determined at
10 μM in both rat and human plasma by equilibrium dialysis and
was considered to be high (95.4ꢀ95.6% bound; Table 11).
Compound 1 was incubated at 2 μM in rat liver microsomes
and hepatocytes in order to determine the in vitro hepatic
’ CONCLUSIONS
A series of novel, selective inhibitors of kinesin spindle protein
have been exemplified. Compounds from this series have been
shown to inhibit tumor cell growth in vitro with a mode of
action that correlates with KSP inhibition. Structureꢀactivity
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Table 12. In Vitro Data Used To Predict in Vivo Clearance
species
in vitro
system
CLint (μmol/min/mg)
(μL/min/106 cells)
predicted total
actual in vivo
(sex)/strain
hepatic CL (mL/min/kg)
% Qh
CL (mL/min/kg)
% Qh
rat (male)/Wistar
microsomes
hepatocytes
<10
<14
<20
22ꢀ48
36
52
4ꢀ13
16ꢀ34
with 0.1% formic acid), using a UV detector at 220, 240, and 254 nm. The
purity of all final compounds was g95%.
Table 13. Pharmacokinetics of Compound 1 (Freebase) after
a Single Iv Bolus Administration
Chiral HPLC separation was carried out by comparison of the elution
times of the compound with a 1:1 mixture of the racemate. The
enantiomeric excess of the final compound was calculated using the
area percent of the UV signal at 220 nm from chiral HPLC analysis.
The rotation parameters were measured by a Perkin-Elmer polarimeter
341. Measurements were made of compounds dissolved at a concentration
of 1 mg/mL in methanol, at 20.0 ꢀC measured at 589 nM.
The synthetic procedures for all compounds mentioned within the
article are described, in extensive detail, in the accompanying Supporting
Information. Final synthetic steps for certain key compounds (namely,
compounds 1, 62, 65, 68 and 71ꢀ78) are outlined below (along with
their respective method numbers).
species
dose
AUCINF
C0
CL
t1/2 Vss
(strain) (mg/kg) (h ng/mL) (ng/mL) (mL/min/kg) (h) (L/kg)
rat (Wistar)
6
2839
1076
36
3.5
10
relationship analysis identified 1 as exhibiting both excellent
enzymatic and cellular potency. This compound exhibited a
favorable pharmacokinetic profile and was progressed into
pharmacodynamic studies in vivo. A rat hollow fiber model has
shown that this compound displays activity consistent with KSP
inhibition causing Colo 205 cells to form monoasters, block in
mitosis and undergo cell death.
These findings supported the selection of 1 as a clinical
candidate for the potential treatment of cancer. The safety,
pharmacokinetics and efficacy of 1 have been assessed in phase
I and II clinical trials in both solid and hematological tumors; the
outcome of one of these trials has recently been reported.44
Method 76. N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]-
thiazolo[5,4-d]pyrimidin-6-yl)propyl]-4-methylbenzamide Hydrochloride.
tert-Butyl N-[3-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyri-
midin-6-yl)propylamino]propyl]carbamate (method 75) (0.117 g,
0.198 mmol) was dissolved in 2 M HCl in ether, and the mixture was
stirred at rt for 20 h. The precipitated product was filtered off and washed
with ether and dried in vacuo to yield the desired product (62) (0.091 g,
87%): 1H NMR (DMSO-d6 300 MHz, 96 ꢀC) δ 7.79 (bs, 3H),
7.37ꢀ6.95 (m, 9H), 5.77 (d, 1H), 5.50 (bs, 1H), 4.83 (d, 1H), 3.36
(t, 2H), 2.72 (s, 3H), 2.46 (t, 2H), 2.39 (s, 3H), 2.20ꢀ2.05 (m, 1H),
1.96ꢀ1.75 (m, 1H), 1.74ꢀ1.40 (m, 2H), 0.63 (t, 3H); m/z 490 (MH+),
C27H31N5O2S 0.5H2O HCl calculated C 60.60, H 6.22, N 13.09, Cl
’ EXPERIMENTAL SECTION
General Information. All solvents used were commercially avail-
able in anhydrous grade. Reagents were utilized without further pur-
ification unless otherwise stated. Organic solutions were dried over
anhydrous sodium sulfate. Evaporation of solvent was carried out using a
rotary evaporator under reduced pressure (600ꢀ4000 Pa (pascals);
4.5ꢀ30 mmHg) with a bath temperature of up to 60 ꢀC. Solvent ratios
are given in volume:volume (v/v) terms.
3
3
6.63, found C 60.59, H 6.30, N 12.88, Cl 6.63.
Method 78. (+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-
[1,2]thiazolo[5,4-d]pyrimidin-6-yl)propyl]-4-methylbenzamide Hydro-
chloride. (+)-tert-Butyl N-[3-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)propyl-(4-methylbenzoyl)amino]propyl]carbamate
(method 77) (0.117 g, 0.19 mmol) was dissolved in 2 M HCl in ether, and
the mixture was stirred at rt for 20 h. The precipitated product was filtered
off and washed with ether and dried in vacuo to yield the pure product (68)
(91 mg, 87%): white powder, mp 127.8ꢀ129.2 ꢀC; 1H NMR (DMSO-d6,
500 MHz, 96 ꢀC) δ 0.63 (t, 3H), 1.40ꢀ1.74 (m, 2H), 1.75ꢀ1.96 (m,
1H), 2.05ꢀ2.20 (m, 1H), 2.39 (s, 3H), 2.46 (t, 2H), 2.72 (s, 3H), 3.36 (t,
2H), 4.83 (d, 1H), 5.50 (bs, 1H), 5.77 (d, 1H), 6.95ꢀ7.37 (m, 9H), 7.79
Temperatures are given in degrees Celsius (ꢀC), and operations were
carried out at room or ambient temperature, that is, at a temperature in
the range of 18ꢀ30 ꢀC.
In general, the course of reactions was followed by thin layer
chromatography or mass spectroscopy and reaction times are given
for illustration only; where a synthesis is described as being analogous to
that described in a previous example, the amounts used are the
millimolar ratio equivalents to those used in the previous example.
NMR data is in the form of delta values for major diagnostic protons,
given in parts per million (ppm) relative to tetramethylsilane (TMS) as
an internal standard, determined at 400 MHz using deuterated chloro-
form (CDCl3) as solvent unless otherwise indicated.
(bs, 3H); m/z 490 (MH+), C27H31N5O2S 1.55HCl calculated C 59.37,
3
H 6.01, N 12.82, found C 59.39, H 6.45, N 12.38.
The following compounds were synthesized according to method 78:
(+)-N-(3-Aminopropyl)-N-[1-[5-[(4-fluorophenyl)methyl]-3-methyl-
4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl]propyl]-4-methylbenzamide
hydrochloride (71): 1H NMR (DMSO-d6, 500 MHz, 96 ꢀC) δ 0.66 (t,
3H), 1.38ꢀ1.74 (m, 2H), 1.82ꢀ1.98 (m, 1H), 2.02ꢀ2.20 (m, 1H), 2.34
(s, 3H), 2.42 (t, 2H), 2.72 (s, 3H), 3.36 (t, 2H), 4.85 (d, 1H), 5.49 (bs,
1H), 5.70 (d, 1H), 7.05ꢀ7.27 (m, 8H), 7.76 (bs, 3H); m/z 508 (MH+),
Analytical mass spectra were run with an electron energy of 70 eV in
the chemical ionization (CI) mode using a direct exposure probe; where
indicated ionization was effected by electron impact (EI), electrospray
(ESP), or atmospheric pressure chemical ionization (APCI); values for
m/z are given; generally, only ions which indicate the parent mass are
reported.
The purity of all final compounds was confirmed by analysis with an
Agilent 1100 liquid chromatography unit (LC) with an Agilent 1100
mass spectroscopy detector (MSD) or a Waters ZQ mass spectrometer
(compounds were detected by mass spectroscopy in APCI+ ionization)
using a Luna 3μ C8 (2) column (100A, 30 ꢁ 2.0 mm), eluting with
mixtures of waterꢀacetonitrile (ranging from 5 to 98% acetonitrile/water
C27H30FN5O2S 3.25HCl calculated C 58.09, H 4.84, N 11.22, found C
3
58.09, H 5.49, N 12.19.
(+)-N-(3-Aminopropyl)-N-[1-[5-[(3-fluorophenyl)methyl]-3-methyl-
4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl]propyl]-4-methylbenzamide
hydrochloride (73): 1H NMR (DMSO-d6, 500 MHz, 100 ꢀC) δ 0.70 (t,
3H), 1.40ꢀ1.54 (m, 1H), 1.62ꢀ1.76 (m, 1H), 1.85ꢀ2.01 (m, 1H), 2.14ꢀ
2.27 (m, 1H), 2.38 (s, 3H), 2.44ꢀ2.49 (m, 2H), 2.76 (s, 3H), 3.35ꢀ3.46
(m, 2H), 4.87 (br s, 1H), 5.48 (br s, 1H), 5.75 (d, 1H), 6.84ꢀ6.96
(m, 2H), 7.06ꢀ7.15 (m, 1H), 7.20ꢀ7.31 (m, 4H), 7.33ꢀ7.41 (m, 1H),
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Figure 4. Compound 1 decreased cell viability, increased cells in mitosis and increased monoaster formation in a rat hollow fiber model. Viable cell
counts (A), the number of monoasters (B), and percent phospho-histone-H3 (PHH3) positive cells (C) obtained from sections of hollow fibers
containing Colo205 cells. Sections were stained with either hematoxylin and eosin or antibodies that recognize α-tubulin and PHH3, respectively. Fibers
were removed from vehicle or compound 1 treated rats 24 and 48 h after administration of a single dose. Statistical analysis was performed using a
standard Student t test (*p < 0.05, **p < 0.01 compared to vehicle at same time point).
Figure 5. Effects of compound 1 on cell viability, monoaster formation and mitosis in hollow fibers. High power fields of view from sections of hollow
fibers showing cell viability at 24 h (A, B) and 48 h (C, D), monoasters at 24 h (E, F), and cells in mitosis at 24 h (G, H). Sections were stained with either
hematoxylin and eosin (AꢀD) or antibodies that recognize α-tubulin (E, F) and phospho-histone-H3 (G, H). Fibers were removed from vehicle (A, C,
E, G) or 1 (B, D, F, H) treated rats 24 h (A, B, EꢀH) or 48 h (C, D) after administration of a single dose. Arrows indicate examples of cells undergoing
cell death (B, D) or monoasters (E, F). Scale bar represents 50 μm.
7.52 (br s, 3H); m/z 508 (MH+), C27H30FN5O2S 3.8HCl calculated C
57.24, H 4.68, N 10.84, found C 57.21, H 5.31, N 12.11.
(+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)propyl]-4-bromobenzamide hydrochloride (75):
1H NMR (DMSO-d6, 500 MHz, 96 ꢀC) δ 0.68 (t, 3H), 1.50ꢀ1.72 (m,
2H), 1.91ꢀ1.96 (m, 1H), 2.13ꢀ2.17 (m, 1H), 2.47 (t, 2H), 2.77 (s, 3H),
3.38 (t, 2H), 4.95 (d, 1H), 5.57 (bs, 1H), 5.80 (d, 1H), 7.13 (m, 2H),
7.28ꢀ7.36 (m, 5H), 7.64 (d, 2H), 7.80 (br, 1H); m/z 554, 556 (MH+),
3
C26H28BrN5O2S 2.8HCl calculated C 52.67, H 4.3, N 10.66, found C
3
52.69, H 5.36, N 9.39.
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Journal of Medicinal Chemistry
ARTICLE
δ 0.52 (d, 3H), 0.94 (d, 3H), 1.15ꢀ1.25 (m, 1H), 1.26ꢀ1.33 (m, 1H),
1.45ꢀ1.58 (m, 1H), 2.32 (m, 2H), 2.38 (s, 3H), 2.78 (s, 3H), 3.32ꢀ3.40
(m, 2H), 5.11 (bd, 1H), 5.56 (bd, 1H), 5.90ꢀ5.93 (d, 1H), 7.11ꢀ
7.38 (m, 8H), 7.58 (b, 2H); m/z 522 (MH+); C28H32FN5O2S 2.3
3
dioxane 2HCl calculated C 56.05, H 6.63, N 8.78, found C 55.42, H
6.45, N 8.77.
3
(+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]-
thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-bromobenzamide
1
hydrochloride (76): H NMR (500 MHz, 90 ꢀC, DMSO-d6) δ 0.48
(d, 3H), 0.93 (m, 3H), 1.10ꢀ1.20 (m, 1H), 1.45ꢀ1.60 (m, 1H),
2.28ꢀ2.41 (t, 2H), 2.63ꢀ2.79 (m, s, 4H), 3.35ꢀ3.43 (m, 2H), 5.08
(m, 1H), 5.62 (m, 1H), 5.96 (d, 1H), 7.30ꢀ7.50 (m, 7H), 7.52ꢀ7.80
(br, m, 4H); m/z 568, 570 (MH+); C27H30BrN5O2S 4HCl calculated C
3
52.11, H 4.23, N 9.8, found C 51.91, H 5.08, N 10.99.
(+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-3-fluoro-4-methylbenzamide
hydrochloride (78): 1H NMR (500 MHz, 90 ꢀC, DMSO-d6) δ 0.48 (d,
3H), 0.93 (d, 3H), 1.18 (m, 1H), 1.53 (m, 1H), 2.32ꢀ2.51 (s, m, 5H),
2.82 (s, 4H), 3.35ꢀ3.43 (m, 2H), 5.10 (m, 1H), 5.62 (m, 1H), 5.94 (d,
1H), 7.11ꢀ7.38 (m, 8H), 7.51 (b, 2H); m/z 522 (MH+); C27H31-
Figure 6. Exposure of compound 1in rat plasma and hollow fibers. Samples
were collected 2 and 24 h after administration of a single intravenous dose.
Plasma drug concentration is shown in black and fiber concentration in white.
(+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)propyl]-3-fluoro-4-methylbenzamide hydrochloride
(77): 1H NMR (DMSO-d6, 500 MHz, 96 ꢀC) δ 0.67 (t, 3H), 1.45 (m,
1H), 1.70 (m, 1H), 1.92 (m, 1H), 2.16 (m, 1H), 2.31 (s, 3H), 2.46 (2H,
hidden by DMSO), 2.76 (s, 3H), 3.39 (t, 2H), 4.93 (d, 1H), 5.54 (bs,
1H), 5.81 (d, 1H), 7.09ꢀ7.52 (m, 8H), 7.74 (br, 3H); m/z 508 (MH+),
N5O2S 3.7HCl calculated C 57.98, H 4.91, N 10.66, found C 58.00, H
3
5.92, N 11.95.
Enzyme IC50 Determination (Eg5 ATPase). The ability of com-
pounds to inhibit Eg5 ATPase activity was examined using a malachite
green assay with recombinant Eg5 (human Eg5 N-terminal 369 amino
acidswith aC-terminal8 histidine tag; 0.4 nM), polymerizedmicrotubules
(0.1 mg/mL) and a nonsaturating concentration of adenosine-50-tripho-
sphate (ATP) (75 μM). In brief, C-terminal His6-tagged recombinant
protein was expressed in bacteria and purified using nickel-nitrilotriace-
tic acid (Ni-NTA) affinity chromatography. Purified protein and
polymerized microtubules in a piperazine-N,N0-bis(2-ethanesulfonic
acid (PIPES) based buffer (pH 6.8) were added to a 384-well plate
containing diluted compound (final 11-point concentration range from
100 μM to 1.7 nM). Reactions were initiated by the addition of enzyme/
microtubule mixture, and plates were allowed to incubate for 1 h at room
temperature (24 ꢀC). Reactions were then quenched with malachite
green reagent and read at A650 after 10 min in a microplate reader. IC50
was determined using XLFit within Activity Base.
Enzyme IC50 Determination (Conventional Kinesin ATPase).
Compound inhibition of conventional kinesin ATPase activity was exam-
ined following the Eg5 malachite green assay protocol with several changes.
A 20 min assay was performed with 3 nM recombinant kinesin (glutathione
S-transferase (GST) tagged human kinesin heavy chain motor protein;
Cytoskeleton catalog no. KR01), 0.6 mg/mL polymerized microtubules
and 300 μM ATP. Compound inhibition of MKLP1 ATPase activity was
also examined following the Eg5 malachite green assay protocol with several
changes. A 20 min assay was performed with 10 nM recombinant MKLP1
(GST-human MKLP1 Kinesin motor domain; Cytoskeleton catalog no.
MP01), 0.15 mg/mL polymerized microtubules and 300 μM ATP.
Cellular IC50 Determination. Colo205 cells were plated in 96-
well flat bottomed plates at a density of 3.3 ꢁ 104 cells per well. After an
initial 24 h period in culture, a predose plate was read to establish
baseline. Remaining cell plates were treated in triplicate with compounds
(using a 9-point concentration range from 10 μM to 0.3 nM + DMSO
control) and left for an additional 72 h. Cell proliferation was evaluated
by the MTS assay, according to the manufacturer’s instructions (Promega,
Madison, WI, p/n G3581; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), in the presence
of phenazine methosulfate (PMS), produces a formazan product that has
an absorbance maximum at 490ꢀ500 nm in phosphate-buffered saline).
Solubility Determination. Equilibrium solubility was determined
in aqueous buffer at pH 7.4. The compounds were serially diluted in 0.1 M
pH 7.4 phosphate buffer at 25 ꢀC with agitation for 24 h. Undissolved
material was removed by filtration, and the filtrates were quantitated
C27H30FN5O2S 3.6HCl calculated C 57.52, H 4.73, N 10.96, found C
3
57.48, H 5.54, N 12.06.
Method 106. N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-
[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide
hydrochloride. tert-Butyl N-[3-[[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-(4-methylbenzoyl)amino]propyl]-
carbamate (method 104) (0.245 g, 0.40 mmol) was dissolved in 4 M HCl in
1,4-dioxane and the mixture was stirred at rt for 20 min. The reaction mixture
was concentrated in a rotary evaporator, and the residue was triturated with
ether. The precipitated product was filtered off, washed with ether and dried
in vacuo to yield N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-
[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide
as the hydrochloride salt (65) (0.219 g, 100%): white powder, mp
1
139ꢀ140 ꢀC; H NMR (DMSO-d6, 300 MHz, 96 ꢀC) δ 0.45 (d, 3H),
0.90 (d, 3H), 1.12ꢀ1.30 (m, 1H), 1.46ꢀ1.63(m, 1H), 2.25 (t, 2H), 2.36(s,
3H), 2.64ꢀ2.7 (m, 1H), 2.68 (s, 3H), 3.34 (t, 2H), 5.06 (d, 1H), 5.59 (d,
1H), 5.90 (d, 1H), 7.20ꢀ7.40 (m, 9H), 7.71 (bs, 3H); m/z 504 (MH+);
C28H33N5O2S 1H2O 1.2HCl, calculated C 59.48, H 6.45, N 12.39, Cl
3
3
7.52, found C 59.61, H 6.51, N 12.36, Cl 7.55.
The following compounds were synthesized according to method 106:
(+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo-
[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide hydrochloride
1
(1): H NMR (500 MHz, 96 ꢀC, DMSO-d6) δ 0.45 (d, 3H), 0.90
(d, 3H), 1.12ꢀ1.30 (m, 1H), 1.46ꢀ1.63 (m, 1H), 2.25 (t, 2H), 2.36 (s,
3H), 2.64ꢀ2.7 (m, 1H), 2.68 (s, 3H), 3.34 (t, 2H), 5.06 (d, 1H), 5.59 (d,
1H), 5.90 (d, 1H), 7.20ꢀ7.40 (m, 9H), 7.71 (bs, 3H); m/z 504 (MH+);
C28H33N5O2S 1H2O 1HCl calculated C 60.26, H 6.50, N 12.55, found
3
3
C 60.20, H 6.28, N 12.40.
(+)-N-(3-Aminopropyl)-N-[1-[5-[(4-fluorophenyl)methyl]-3-methyl-
4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl]-2-methylpropyl]-4-methyl-
benzamide hydrochloride (72): 1H NMR (500 MHz, 90 ꢀC, DMSO-d6)
δ 0.47 (d, 3H), 0.92 (d, 3H), 1.10ꢀ1.28 (m, 1H), 1.44ꢀ1.56 (m, 1H),
2.27 (t, 2H), 2.36 (s, 3H), 2.66ꢀ2.72 (m, 1H), 2.75 (s, 3H), 3.35 (t, 2H),
5.04 (d, 1H), 5.57 (d, 1H), 5.86 (d, 1H), 7.12ꢀ7.43 (m, 8H), 7.71ꢀ7.81
(m, 3H); m/z 522 (MH+); C28H32FN5O2S 4HCl calculated C 57.57, H
3
4.83, N 10.49, found C 57.53, H 5.70, N 11.22.
(+)-N-(3-Aminopropyl)-N-[1-[5-[(3-fluorophenyl)methyl]-3-methyl-
4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl]-2-methylpropyl]-4-methyl-
benzamide hydrochloride (74): 1H NMR (500 MHz, 90 ꢀC, DMSO-d6)
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Journal of Medicinal Chemistry
ARTICLE
against a standard in DMSO prepared from the same DMSO stock using a
generic HPLCꢀUV method with mass spectroscopy mass confirmation.
Animals. Hans Wistar rats were purchased from Charles River. All
procedures were conducted in accordance with the Institute for Labora-
tory Animal Research Guide for the Care and Use of Laboratory Animals
and within the protocols approved by the Institute of Animal Care and
Use Committee at AstraZeneca.
Present Addresses
Lilly Corporate Centre, Indianapolis, Indiana 46285, United States.
^Memorial Sloan-Kettering Cancer Centre, 1272 York Avenue,
New York, New York 10065, United States.
Notes
§Previously AstraZeneca Boston.
#Previous employees of Organix Inc., 240 Salem St., Woburn,
Massachusetts 01801, United States.
Rat Hollow Fiber Model. Fibers made from polyvinylidene
difluoride (molecular weight cutoff, 500 kDa; Spectrum) were immersed
in 70% ethanol for 72 h, flushed with distilled water, and autoclaved.
Colo205 cells (obtained from American Type Culture Collection) were
suspended in Roswell Park Memorial Institute (RPMI) medium with
20% fetal bovine serum and loaded into the fibers (5 ꢁ 106 cells/mL)
and the fibers sealed at both ends. Following overnight incubation in
media at 37 ꢀC, fibers were implanted subcutaneously into rats. After
several hours, a single dose of 1 or vehicle (sterile saline) was admin-
istered over approximately 1 min through a jugular vein cannula (3 rats
per treatment group). Fibers and plasma were collected at 2, 24, or 48 h
after treatment and processed for either histology/immunohistochem-
istry (IHC) or drug exposure analysis (1 fiber per time-point per assay).
Histology/Immunohistochemistry (IHC). Formalin fixed par-
affin embedded sections were prepared from the fibers and placed onto
slides. Hematoxylin and eosin staining was performed on a Sakura 2000
autostainer. In brief, the slides were deparaffinized, hydrated, stained in
Gill #2 hematoxylin (Polysciences Inc.), rinsed in water, blued in Lerner
Bluing reagent (Fisher) and counterstained in Eosin Y (Shandon).
Alpha tubulin and PHH3 immunostaining were performed on a Ventana
Discovery XT Immunostainer. Slides were incubated in the primary
PHH3 antibody (CST-9601, Cell Signaling Technology) at a 1:50
dilution followed by detection with a biotinylated goat anti-rabbit
secondary antibody (PK-6101, Vector Laboratories). Slides were in-
cubated in the primary α-tubulin antibody (Sigma T9026) at a 1:20,000
dilution followed by detection with a biotinylated mouse secondary
antibody (Vector, PK-6102). Secondary antibodies were detected with a
Ventana DABMap kit (Ventana cat. no. 760-124; (DAB refers to 3,30-
diaminobenzidine)). Slides were then counterstained with hematoxylin
(Ventana 760-2021) and blued with bluing reagent (Ventana 760-2037)
before being dehydrated, cleared and mounted.
’ ACKNOWLEDGMENT
We thank Nancy DeGrace for determining chiral separation
conditions for all compounds mentioned.
’ ABBREVIATIONS USED
APCI, atmospheric pressure chemical ionization; ATP, adeno-
sine-50-triphosphate;CDCl3, deuterated chloroform; CI, chemical
ionization; CYP450, cytochrome P450; DAB, 3,30-diaminoben-
zidine; EI, electron impact; ESP, electrospray; GST, glutathione
S-transferase; HsEg5, Homo sapiens Eg5; IHC, immunohisto-
chemistry; iv, intravenous; KSP, kinesin spindle protein; LC,
liquid chromatography; LCK, lymphocyte specific protein tyrosine
kinase; MAPKAP-K1a, mitogen-activated protein kinase-acti-
vated protein kinase 1a; MKLP, mitotic kinesin-like protein;
MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium; MSD, mass spectroscopy
detector; Ni-NTA, nickel-nitrilotriacetic acid; PHH3, phospho-
histone-H3; PIPES, piperazine-N,N0-bis(2-ethanesulfonic acid);
PMS, phenazine methosulfate; RPMI, Roswell Park Memorial
Institute; TMS, tetramethylsilane
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