Scholarly article on N-hydroxy 2-{6-[(6-fluoroquinolin-2-ylmethyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxamide 1256448-47-1 from Journal of Medicinal Chemistry p. 8663

J. Med. Chem. 2010, 53, 8663–8678 8663

DOI: 10.1021/jm101177s

Discovery of 2-(6-{[(6-Fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-3-yl)-

N-hydroxypyrimidine-5-carboxamide (CHR-3996), a Class I Selective Orally

Active Histone Deacetylase Inhibitor

David Moffat,*^,†Sanjay Patel,^†Francesca Day,^†Andrew Belfield,^†Alastair Donald,^†Martin Rowlands,^‡Judata Wibawa,^‡

Deborah Brotherton,^†Lindsay Stimson,^‡Vanessa Clark,^†Jo Owen,^†Lindsay Bawden,^†Gary Box,^‡Elisabeth Bone,^†

Paul Mortenson,^§Anthea Hardcastle,^‡Sandra van Meurs,^†Suzanne Eccles,^‡Florence Raynaud,^‡and Wynne Aherne^‡

^†Chroma Therapeutics Ltd., 93 Milton Park, Abingdon, OX14 4RY, U.K., ^‡Haddow Laboratories, Cancer Research UK, Centre for Cancer

Therapeutics, The Institute for Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey, SM2 5NG, U.K., and ^§Evotec (UK) Ltd.,

114 Milton Park, Abingdon, OX14 4SA, U.K.

Received September 9, 2010

A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavail-

ability is described. The compounds are potent enzyme inhibitors (IC₅₀values less than 100 nM), and

improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA)

through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice,

comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in

human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r

(CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo

xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro

studies. On the basis of these results, 21r was nominated for clinical development.

Introduction

rangeof tumor types either alone orin combinationwithother

therapies.⁸

Histone deacetylases (HDACs^a) are a family of epigenetic

regulator enzymes, the inhibition of which leads to a reversal

of tumor suppressor silencing, cell cycle arrest, differentiation,

and/or apoptosis.¹Because of these effects, which manifest

themselves most strikingly in transformed cells, HDAC in-

hibition has emerged as a strategy to reverse aberrant epige-

netic changes associated with cancer.^2,3A number of HDAC

inhibitors have shown antitumor activity in animal models of

human cancer and are now in clinical trials (Figure 1).⁴The

first such agent to reach pivotal clinical trials was the hydro-

xamate, SAHA^5-7(vorinostat); this drug has been approved

for use in the treatment of cutaneous T-cell lymphoma. The

cyclic depsipeptide romidepsin has been registered for the

treatment of the same disease. A wealth of preclinical data

suggest that HDAC inhibitors have potential against a wide

The HDACs comprise three separate classes of enzyme.⁹

Both class I and class II enzymes are metalloenzymes containing

a catalytic zinc atom. There are thought to be at least 11

isoforms of the metalloenzyme HDAC proteins: class I HDACs

(HDACs 1, 2, 3, and 8) are ubiquitously expressed and generally

detected in the nucleus, while class II HDACs (HDACs 4, 5, 6, 7,

9, and 10) can shuttle between the cytoplasm and nucleus.

HDAC11 has a related catalytic domain but has only weak

homology with the others. The class III enzymes are not

metalloenzymes and, while relevant to certain human diseases,

are not yet strongly implicated in cancer.

The major objectives of our discovery efforts toward

therapeutically useful HDAC inhibitors were to achieve good

intratumoral distribution of drug via oral administration and

class I isoform selectivity. The latter goal was driven by the

hypothesis that these isoforms are those most predominantly

involved in cellular proliferation.¹⁰It has been suggested from

extensive analysis of published protein and mRNA expression

of HDAC isoforms that class I HDACs are often over-

expressed in tumors compared to corresponding normal

tissue, and this overexpression generally correlates with poor

prognosis. In contrast, expression of class II HDACs may be

associated with better prognosis, suggesting that their inhibi-

tion may not be useful in cancer therapy.¹¹

*To whom correspondence should be addressed. Phone:

þ44(0)1235 829120. Fax: þ44(0)1235 828125. E-mail: david.moffat@

chromatherapeutics.com.

^aAbbreviations: HDAC, histone deacetylase; PSA, polar surface

area; SAHA, suberoylanilide hydroxamic acid; HDACi, histone deace-

tylase inhibitor; TBME, tert-butyl methyl ether; TFA, trifluoroacetic

acid; DMAP, 4-dimethylaminopyridine; EDC, 1-ethyl-3-(3-dimethyla-

minopropyl)carbodiimide; HOBt, hydroxybenzotriazole; mCPBA, m-

chloroperoxybenzoic acid; ZBG, zinc binding group; AUC, area under

curve; Cl, clearance; F, oral bioavailability; HLM, human liver micro-

somes; MLM, mouse liver microsomes; BOC, tert-butoxycarbonyl;

Boc₂O, di-tert-butyl dicarbonate; EtOAc, ethyl acetate; THF, tetrahy-

drofuran; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EGF,

epidermal growth factor; PBS, phosphate buffered saline; FACS, fluor-

escence-activated cell sorting; ND, not determined; ELISA, enzyme-

linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate

dehydrogenase; PARP, poly ADP-ribose polymerase.

Chemistry

We designed a series of N-hydroxypyrimidine-5-carboxa-

mides as potential HDAC inhibitors based on an analysis of

all previously described families that assessed their ability to

r

2010 American Chemical Society

Published on Web 11/16/2010

pubs.acs.org/jmc

8664 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

Figure 1. HDAC inhibitors under clinical investigation.

meet our goal of robust physicochemical properties to facil-

itate improved cell permeability and oral dosing as well as to

provide novelty. We decided to utilize the pyrimidine hydro-

xamate moiety as the zinc-binding group (ZBG) in our

approach, as this seemed likely to be the most metabolically

stable functionality that would be compatible with good

HDAC inhibitory activity. The Johnson & Johnson group

has previously reported on HDAC inhibitors from this chem-

ical class.^12-14In parallel to our work,¹⁵this group has

pursued a strategy aimed at improving the pharmacodynamic

properties of their initial lead series through introduction of

an ionizable group to improve solubility.¹⁶

Work on the project was initiated with docking studies

using an available structure of the class I isoform HDAC8¹⁷

based on coordination of the pyrimidine hydroxamate unit to

the zinc ion at the base of the catalytic site. We believed that

this wouldpermit the identification of novel linker groups that

would appropriately position the inhibitor headgroup in the

hydrophobic region at the entrance of the active site. These

studies indicated that the hexahydropyrrolo[3,4-c]pyrrole and

azabicyclo[3,1,0]hexane linkers (Figure 2) were of a requisite

length to provide good interaction between aryl- or heteroaryl

substituents and this hydrophobic surface. We have pre-

viously described the optimization of the hexahydropyrrolo-

[3,4-c]pyrrole linked compounds.¹⁸In this article we detail the

development of the azabicyclo[3,1,0]hexane series toward a

compound that has progressed to clinical study.

Our approach to the synthesis of the pyrimidine hydroxamate

target molecules was dependent on the availability of several key

intermediates (Scheme 1). Intermediate 2 was prepared from

commercially available ethyl 4-chloro-2-(methylsulfanyl)pyri-

midine-5-carboxylate in a two step procedure involving zinc

mediated removal of the 4-chloro substituent followed by

subsequent oxidation of the 2-methanesulfanyl group. The

route to our linker unit required the synthesis of the Boc-

protected 6-amino-3-aza-bicyclo[3.1.0]hexane 8 following a

multistep procedure starting from N-benzylmaleimide via the

known intermediate 6.¹⁹Selenium dioxide mediated oxidation

of 6-fluoro-2-methylquinoline gave the aldehyde intermediate

Figure 2. Structural components of pyrimidine hydroxamate HDAC

inhibitors containing hexahydropyrrolo[3,4-c]pyrrole (A) and azabi-

cyclo[3,1,0]hexane (B) linkers.

10 in excellent yield. The differentially Boc-protected methyl-

aminoazabicyclohexane derivative 12 was available from a

previously described method,²⁰as was O-(1-isobutoxyethyl)-

hydroxylamine 11.²¹This was employed to introduce the

hydroxamate functionality in the final steps of our analogue

preparation.

Sulfonation of the amino group of compound 8 with aryl-

or heteroarylsulfonyl chlorides followed by removal of the

Boc group gave suitable intermediates 13a,b for condensation

with the pyrimidine intermediate 2 through displacement of the

2-methanesulfonyl substituent (Scheme 2). Base hydrolysis of

the intermediate ester gave carboxylic acids 14a,b which were

coupled with the protected hydroxylamine 11 to give intermedi-

ates that could be purified via flash chromatography on silica.

Subsequent deprotection of the hydroxamate was facile under

acidic conditions, giving target molecules 15a and 15b. The

corresponding amide analogues 18a-cwere prepared by similar

methodology beginning with amidation of 8 with readily avail-

able acid chlorides. In the case of the alkylamine analogues

21a-r the six-step sequence from 8 proved to be a robust and

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8665

Scheme 1. Preparation of Intermediates^a

efficient process in which the intermediates 19 and 20 could

generally be reached without the need for a purification step.

The conformation of the azabicyclo[3,1,0]hexane linker unit

was determined by X-ray diffraction studies on compound

21r. The constrained three-membered ring was observed in the

exo-conformation, consistent with previous reports.^19,22

Methylation of the intermediate sulfonamide, formed by

the reaction of amine 8 and 2-naphthylenesulfonyl chloride

provided 22, facilitating the synthesis of final hydroxamate 23

(Scheme 3). The condensation of 8 with 1-chloroisoquinoline

furnished the desired aminoisoquinoline intermediate 24,

allowing access to final compound 25. The extended methyl-

amino analogue 27 was obtained by the reaction of Boc-

protected azabicyclo[3,1,0]hexane 12 with the 2-methanesul-

fonyl intermediate 2 followed by subsequent ester hydrolysis

to give intermediate 26. The hydroxamate functionality of 27

was introduced as for previous analogues.

Results and Discussion

The intrinsic HDAC potency of test compounds was

established through IC₅₀determination (Table 1) in an assay

utilizing nuclear extract from HeLa cells. Similar levels of

inhibition were observed for the sulfonamide 15a, amide 18a,

and alkylamino 21a compounds, confirming a hypothesis

from our modeling studies that all these linkers from the

azabicyclohexane spacer would place the 2-naphthyl head-

group in a similar position at the surface of the protein (Figure 3).

However, wedidobservethat the cell-to-enzyme potency ratio

(GI₅₀(HCT116)/IC₅₀(HDAC)) was reduced for the naphthyl-

methylene analogue 21a when growth inhibition of the

HCT116 human colon carcinoma cell line was examined.

The cell-to-enzyme ratios for 15a, 18a, and 21a were 73, 32,

and 13, respectively. We attributed this observation to the

lower calculated PSA of 21a in comparison to 15a and 18a.

This hypothesis appears to be supported by an analysis of an

expanded panel of 84 pyrimidine hydroxamates made in this

project. Although the absolute correlation between PSA

and cell-to-enzyme ratio is relatively weak, the trends for

^aReagents and conditions: (a) Zn powder, H₂O, t-BuOH, THF, 85 °C,

57%. (b) mCPBA, THF, 0 °C, 77%. (c) BrCH₂NO₂, K₂CO₃, CH₃CN,

room temp, 42%. (d) NaBH₄, BF₃THF, 45-50 °C, 42%. (e) CH₃CHClO-

3

COCl, ClCH₂CH₂Cl, 55-65 °C, 25%. (f) Boc₂O, DMAP, CH₂Cl₂, room

temp, 92%. (g) 10% Pd-C, H₂, EtOH, roomtemp, 95%. (h) SeO₂, dioxane,

100 °C, 87%.

Scheme 2. Preparation of Pyrimidine Hydroxamate Analogues^a

^aReagents and conditions: (a) RSO₂Cl, triethylamine, CH₂Cl₂, 0 °C to room temp. (b) TFA, CH₂Cl₂, room temp. (c) Ethyl 2-(methylsulfonyl)pyrimidine-

5-carboxylate 2, K₂CO₃, CH₃CN, room temp. (d) NaOH, MeOH, THF, room temp. (e) O-(1-Isobutoxyethyl)hydroxylamine, EDC hydrochoride, HOBt,

triethylamine, room temp. (f) TFA, CH₂Cl₂, room temp or 4 M HCl in dioxane, CH₂Cl₂, room temp. (g) ROCl, pyridine, 0 °C. (h) 4 M HCl in dioxane, CH₂Cl₂,

room temp. (i) RCHO, NaBH₄, MeOH, NaOH, room temp, 58%. (j) 4 M HCl in dioxane, CH₂Cl₂, room temp.

8666 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

Scheme 3. Preparation of Further Pyrimidine Hydroxamate Analogues^a

^aReagents and conditions: (a) 2-(tetrahydro-2H-pyran-2-yl)hydroxylamine, EDC hydrochloride, HOBt, DMF, room temp, 90%. (b) NaH, (MeO)₂SO₂,

THF, room temp. (c) TFA, CH₂Cl₂, MeOH, room temp, 3% over two steps. (d) 1-Chloroisoquinoline, 100 °C. (e) Ethyl 2-(methylsulfonyl)pyrimidine-

5-carboxylate 2, K₂CO₃, CH₃CN, DMF, room temp. (f) NaOH, THF, H₂O, room temp, 30% over three steps. (g) O-(1-Isobutoxyethyl)hydroxylamine, EDC

hydrochloride, HOBt, triethylamine, room temp, 36%. (h) 4 M HCl in dioxane, room temp, 58%. (i) Ethyl 2-(methylsulfonyl)pyrimidine-5-carboxylate 2,

K₂CO₃, CH₃CN, DMF, room temp, 90%. (j) 4 M HCl in dioxane, CH₂Cl₂, room temp, 97%. (k) 2-Naphthaldehyde, NaBH₄, MeOH, room temp. (l) NaOH,

THF, H₂O, room temp. (m) O-(1-Isobutoxyethyl)hydroxylamine, EDC hydrochloride, HOBt, triethylamine, room temp, 46% over two steps. (n) 4 M HCl in

dioxane, CH₂Cl₂, room temp, 50%.

low cell-to-enzyme ratios, arbitrarily set at <20, were made

evident by binning PSA data (Figure 4). For 53 compounds

that had cell-to-enzyme ratio of less than 20 (Figure 4a), 58%

potency against the target, in relation to the aminoalkyl

analogues 21a-r. The introduction of an additional methy-

lene unit in compound 27 led to an HDAC IC₅₀value of

32 nM which compared favorably to the potency of 21a which

inhibits the enzyme with a potency of 14 nM. Thus, it would

appear that HDAC activity is relatively insensitive to linker

length in this series. It has been reported that, at least for

HDAC8, the protein surface at the opening to the active site is

malleable allowing it to accommodate binding of a variety of

ligands.²⁴Thus, the narrow range of inhibition within our

series is unsurprising.

Pharmacokinetic Data. With good enzyme and cell po-

tency demonstrated, we decided to investigate the exposure

of this series of compounds by the oral route of administra-

tion to ascertain which compounds to progress to efficacy

studies. With many compounds of similar potency available,

we pursued a triaging strategy based on comparison of the

AUCs of these analogues following oral cassette dosing at 3

mg/kg in mice, the best compounds being shown in Table 3.

Interestingly 21r had significantly higher exposure in the

spleen in comparison to the 2-naphthyl analogue 21a. This

presumably reflects the ability of the fluoroisoquinoline

group in 21r to prevent the rapid oxidative metabolism of

the naphthyl headgroup observed for 21a in microsomal

studies (data not shown). From this work 21f and 21r were

identified as lead compounds for progression.

2

˚

had a PSA value of less than 100 A . In comparison, for the 31

compounds with a ratio greater than 20, 81% of compounds

2

had a PSA of >100 A (Figure 4b), suggesting that targeting a

˚

lower PSA value was important for good cell activity. Since

these lower PSA values are achieved by the introduction of the

alkylamino linkage, we decided to focus our optimization on

these derivatives. We also believed that the more lipophilic

nature of these analogues resulting from reduced PSA in

conjunction with the introduction of the ionizable aminoalkyl

linker would perhaps lead to improved distribution of drug to

the tumor.²³Expansion of the SAR of compound 21a in-

dicated that many aryl and heteroaryl groups are tolerated as

the aminoalkyl substituent and that good cell-to-enzyme

ratios are maintained.

An additional factor limiting cell penetration of 15a may

have been the ionization of the sulfonamide linker. The

calculated pK_afor this group is 3.4, suggesting that it will be

negatively charged at physiological pH. We therefore decided

to investigate whether methylation of the sulfonamide nitro-

gen would mitigate this effect. The HDAC IC₅₀value of

23 nM for 23 (Table 2) indicated that while the introduction

of the methyl substituent was not detrimental to potency,

activityinthe cellgrowthinhibition assay was stillmoderateat

1 μM. This result enhanced our belief that high PSA limits cell

penetration of these compounds.

Further in vitro profiling of 21f and 21r indicated that both

compounds demonstrated moderate to good permeability in a

Caco-2 monolayer assay, particularly in comparison to the

prototypical sulfonamide inhibitor 15a (Table 4). Good meta-

bolic stability in mouse and human liver microsomes was also

We next decided to examine the effect of linker length on

potency through the synthesis of compounds 25 and 27. The

truncated aminoisoquinoline compound 25 maintained good

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8667

Table 1. Inhibition of HDAC Activity IC₅₀, Growth Inhibition GI₅₀

Values in Human Colorectal Cancer Cell Line HCT116, and PSA Values

for Compounds 15a,b, 18a-c, and 21a-r

Figure 3. Proposed binding mode of 15a with HDAC8 (cross section).

The Zn^2þion is shown as a sphere and the protein as a surface.

Figure 4. Pie charts showing the percentage distribution of pyrimi-

dine hydroxamate compounds in PSA bins. Compounds are divided

between those with cell-to-enzyme ratio of <20 (a) and those with cell-

2

˚

to-enzyme ratio of >20 (b). Compounds with PSA of 70-100 A are

2

˚

represented in bright green, 100-130 A in light green, 130-160 A in

2

˚

amber, and 160-190 A in red. Pie charts were generated with Vortex

(Dotmatics Ltd., U.K.).

Table 2. Inhibition of HDAC Activity (IC₅₀), Growth Inhibition (GI₅₀)

in Human Colorectal Tumour Cell Line HCT116, and PSA Values for

Compounds 23, 25, and 27

HDAC

IC₅₀(nM)^a

HCT116

GI₅₀(nM)^b

cell/enzyme

ratio^c

PSA

2

(A )

˚

compd

23

25

27

23

18

32

1000

130

44

7

124

103

90

370

12

^aValues are the mean of two independent determinations. SAHA was

used as a positive control: IC₅₀= 87 ( 13 nM. ^bn = 6. SAHA gave a

GI₅₀(mean of three experiments) in these assays of 920 ( 120 nM. ^cCell/

enzyme ratio = (HCT116 GI₅₀)/(HDAC IC₅₀).

^aValues are the mean of two independent determinations or mean (

SD of three or more experiments. The K_ifor 21r was 4.8 nM. SAHA was

used as a positive control: IC₅₀= 87 ( 13 nM. ^bn = 6. SAHA gave a

GI₅₀(mean of three experiments) in these assays of 920 ( 120 nM. ^cCell/

enzyme ratio = (HCT116 GI₅₀)/(HDAC IC₅₀). ^dNot determined.

amide and carboxylic acid being observed (Figure 5). No

detectable metabolites were identified from human micro-

somes. Compounds 21f and 21r also displayed favorable

drug-drug interaction profiles, with no significant inhibitory

activity being observed against CYP2C19, CYP2D6, and

CYP3A4 at concentrations below 10 μM. The equilibrium

solubility²⁵of 21a and 21r was measured in phosphate buffer

observed, particularly for 21r, with a significant amount of

parent drug remaining after 30 min of incubation in both

preparations. The glucoronide was the major metabolite iden-

tified in mouse liver microsomes with smaller amounts of the

8668 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

at pH 5.5 and 7.4. The solubility of 21a was pH dependent as

expected, but the solubility of 21r was surprisingly poor,

especially at the lower pH. Despite this, we were encouraged

to profile the in vivo PK of 21f and 21r in other species to

further assess their suitability as potential drug candidates. We

were pleased to find that the oral exposure demonstrated in the

mouse oral PK studies translated into good bioavailability for

both compounds in the dog, with F values of 56% and 40% for

21f and 21r, respectively. In the rat, 21r also demonstrated good

bioavailability (F = 27%); 21f was not tested (Table 5).

Pharmacology. We were keen to determine the HDAC

isoform selectivity of 21f and 21r, and so their IC₅₀values

against a panel of HDAC isoforms were determined (Table 6).

We were pleased to observe that 21f and 21r showed excellent

potency against the class I isoforms, HDACs 1, 2, and 3, and

good selectivity over HDACs 5 and 6, members of the class II

family. These two compounds were ∼700-fold selective for

HDAC1 compared to HDAC6, whereas SAHA showed

equipotent activity. To further investigate this subtype selec-

tivity at the cellular level, we used the HCT116 cell line to

evaluate the acetylation status of histone H3 in comparison

to that of the HDAC6-specific substrate R-tubulin. We

found that 21f, 21r, and SAHA have almost equivalent

effects on histone acetylation at equitoxic doses whereas

SAHA has a greater effect on tubulin acetylation (Figure 6).

The inability of 21f and 21r to induce significant acety-

lation of R-tubulin confirmed that they are not effective

inhibitors of HDAC6. The cleavage of PARP was also

observed in HCT116 cells (Figure 7), this being indicative of

apoptosis.

synergistic manner with other antitumor agents in a number

of in vitro studies.²⁷Therefore, conventional Chou and

Talalay median effect analysis²⁸was conducted following

the generation of appropriate cell proliferation data using

21f and 21r in combination with erlotinib²⁹(EGF receptor

kinase inhibitor), decitabine³⁰(DNA methyltransferase in-

hibitor), and tosedostat³¹(aminopeptidase inhibitor). As

indicated by the combination index (CI),³²good additivity

and synergy were seen across a panel of human cancer cell

lines (Table 8). The most striking synergy was observed with

a combination of 21r and tosedostat against cells originating

from pancreatic carcinoma and malignant melanoma.

Flow cytometric analysis was employed to determine the

relative abilities of 21f, 21r, and SAHA to induce cell cycle

arrest (Table 9), following the treatment of HCT116 cells

with 300 nM inhibitor for 15 h. An increase in cells in the sub-

G1 phase was observed for all three compounds, but only 21r

Given the important role of HDACs in tumor cell pro-

liferation, we measured the antiproliferative effects of 21f

and 21r, again in comparison to SAHA, against an expanded

panel of transformed human cell lines originating from both

solid and hematological cancers (Table 7). As shown, 21r

exhibits growth inhibition in the range 31-750 nM and was

generally 2-3 times more potent than 21f across the cell

panel. In comparison to SAHA, 21r was 5-20 times more

potent.

With attention turning increasingly toward combination

trials of HDAC inhibitors²⁶we decided to investigate the

synergy between our lead compounds with other anticancer

agents. HDAC inhibitors have been reported to act in a

Figure 5. Metabolism of 21f and 21r in the mouse.

Table 5. Pharmacokinetic Parameters of Compounds 21f and 21r in

Rat^aand Dog^b

iv (1 mg/kg)

AUC_0-tCl

po (10 mg/kg)

AUC_0-t

(ng h/mL) ((mL/min)/kg) (ng h/mL) t_1/2(h) F (%)

3

dog 21f

dog 21r

rat 21r

265

241

124

63

70

1475

950

102

ND^c

0.9

56

40

27

134

5.2

^aThe vehicle for rat iv and po dosing was 1% DMSO-HPβC (11.25%

w/v). ^bThe vehicle for dog iv dosing was 5% DMSO-HPβC (11.25% w/v)

and 5% DMSO-saline for the po arm. ^cNot determined.

Table 3. AUC Values from Mouse po Cassette Studies^a

Table 6. Inhibition of HDAC Isoforms (IC₅₀in nM)^a

AUC_0-t(ng h/mL)

3

compd

plasma

spleen

spleen/plasma ratio

HDAC

21f

21r

SAHA

21a

21f

21k

21o

21q

21r

50

58

83

52

42

93

145

436

251

219

294

775

2.9

7.5

3.0

4.2

7.0

8.3

1

2

3

5

6

3

30

24

14

10

4

7

150

3000

30

500

3300

200

2100

^aAssays were performed by BPS Biosciences (San Diego, CA) using

human recombinant enzyme.

^aCassettes comprised four compounds dosed at 3 mg/kg po to CD1 mice.

Table 4. In Vitro ADME Properties of HDAC Inhibitors

Caco-2 P_app(cm/s)

in vitro Cl (% remaining 30 min)

CYP IC₅₀(μM)

solubility (μg/mL)

pH 5.5 pH 7.4

compd

A2B

B2A

MLM^a

HLM^b

2C19

2D6

3A4

15a

21a

21f

21r

<0.5

3

8.9

20

27

17

66

43

40

38

21

10-50

>50

50

10-50

>50

10-50

>50

74

34

100

43

208

ND^c

3

12

24

9

10-50

>50

ND^c

1

31

>50

10-50

^aMLM = mouse liver microsome. ^bHLM = human liver microsome. ^cNot determined.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8669

tumor growth models were consistent with HDAC inhibition.

Female CrTac:NCr-Fox1(nu) mice with established HCT116

tumor xenografts were dosed orally on a daily schedule for

16 days with 50 mg/kg 21f. Although the efficacy of 21f in this

study was disappointing (Figure 8a), the compound was well

tolerated and an increase in histone acetylation was observed in

the tumors of 21f-treated animals relative to vehicle animals

for at least 4 h (Figure 8b). As expected from in vitro studies,

R-tubulin acetylation was unchanged in the tumors from

21f-treated mice compared with controls. Concentrations of

21f in the spleen were 4-14 times higher than those in plasma,

consistent with our earlier cassette PK study (Table 3). The

measured tumor concentration of 21f was sustained for at least

4 h at levels more than 4 times greater than its IC₅₀, consistent

with the observed duration of increased histone acetylation.

In a second HCT-116 mouse xenograft study (Figure 9a)

we compared the efficacy of 21f (75 mg/kg po q.d.) with 21r

(50 mg/kg po q.d.). We found that 21r, even at a lower dose

than 21f, resulted in almost complete suppression of tumor

growth. Examination of tumor concentrations revealed that

21r (133 ng/mL) achieved levels almost 5 times higher in

comparison to the higher dose of 21f (28 ng/mL). The ratio of

tumor to plasma concentrations for 21r (Figure 9b) also

reflected our earlier studies of distribution to spleen

(Table 3). Given that 21r in general showed greater in vitro

potency and enhanced distribution to tumor compared to

21f, this result was perhaps unsurprising. We therefore

decided to examine the efficacy of 21r in a second human

tumor xenograft model. A study was performed to evaluate

the effect of a lower dose level of 21r on subcutaneous growth

of a LoVo human colorectal xenograft in female BALB/c

nude mice (Figure 10). The compound was administered

orally at 50 and 25 mg/kg daily, with 21r again substantially

reducing tumor volume and demonstrating an apparent dose

dependency. As a result of these observations 21r (CHR-

3996)¹⁵was selected as our clinical candidate.

Figure 6. Substrate selectivity of 21f and 21r in HCT116 cells.

Human HCT116 colon carcinoma cells were incubated with the

indicated concentrations of 21f, 21r, and SAHA for 30 h. Total cell

lysates were prepared and analyzed by SDS-PAGE and Western

blotting using antibodies to acetylated H3K9 (Abcam 1:5000 dilution)

and acetylated R-tubulin (Abcam, 1:500 dilution). GAPDH was used as

a loading control. Similar results were obtained using a fixed cell ELISA

(see Supporting Information).

Figure 7. Induction of PARP cleavage by 21f and 21r in comparison to

SAHA. Human HCT-116 colon carcinoma cells were incubated with

the indicated concentrations of 21f, 21r, and SAHA for 30 h. Total cell

lysates were prepared and analyzed by SDS-PAGE and Western

blotting using antibodies to PARP and cleaved PARP (both Cell

Signaling, 1:1000 dilution). GAPDH was used as a loading control.

Conclusion

Table 7. Antiproliferative Activity of 21f and 21r in a Panel of Human

Tumour and Leukemic Cell Lines (GI₅₀in nM)^a

Throughanoptimization processwehaveidentified21r asa

7 nM inhibitor of HDAC with a class I isotype selectivity that

shows good potency for the inhibition of tumor cell growth.

We have demonstrated that the compound can be delivered via

oral administration with good distribution to tissue as mea-

sured by drug level in the mouse spleen and xenograft tissue

and that the compound shows completeinhibition of growth in

two human tumor xenograft models. 21r has been selected for

a phase 1 clinical trial and represents a highly promising new

agent with the potential to be applied to a broad range of

human cancers.

cell line

HCT116

LoVo

origin

colon carcinoma

21f 21r SAHA

210 72 740

310 140 1700

410 210 3150

A2780

Du145

PC3

ovarian adenocarcinoma

prostate adenocarcinoma

92 48 1050

260 160 2000

HepG2

MG63

MiaPaca-2

A549

hepatocellular adenocarcinoma 470 230 4600

osteocarcinoma

1600 750 >10 μM

620 280 3500

500 250 5700

210 105 1100

1100 560 3300

pancreatic carcinoma

lung adenocarcinoma

large cell lung carcinoma

COR-L23

COR-L23 “R” doxorubicin resistant variant

Experimental Section

Molt-4

Hut78

U937

acute lymphoblastic leukemia

T-cell lymphoma

histiocytic lymphoma

71

60

31 670

31 295

Biological Methods. Measurement of HDAC Activity. The

HeLa cell nuclear extract (catalog no. CC-01-20-50) was

obtained from Cilbiotech S.A., Mons, Belgium. Histone deace-

tylase (HDAC) activity was determined using the Fluor De Lys

system with the substrate (catalog no. KI-104) and the developer

concentrate (catalog no. KI-105) purchased from Biomol Inter-

national, Palatine House, Matford Court, Exeter, U.K. All

assays were run in polystyrene, flat-bottomed Immulon 2HB

96-well plates purchased from Thermo Life Sciences, Milford,

MA, U.S. The fluorimetric assay of HDAC activity was carried

out as described in the Biomol technical manual. The Fluor De

Lys substrate, developer concentrate, and HeLa cell nuclear

extract were all diluted with the assay buffer (25 mM Tris-HCl,

120 52 450

ND^b110 2100

MRC5- SV 2 transformed lung fibroblast

^an = 6 replicates measured by WST-1 assay. ^bNot determined.

showed accumulation at G2/M compared to nontreated cells

at this concentration and time-point.

The promising pharmacological and PK profiles of 21f

and 21r prompted an in vivo efficacy study of these com-

pounds against some of the human cancer cell lines employed

in our in vitro cell growth inhibition studies. Initially we

wanted to ensure that pharmacodynamic effects observed in

8670 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

Table 8. In Vitro Synergy Studies between HDAC Inhibitors and Anticancer Agents

combination

schedule

cell line

H358

type of cancer

NSCLC

CI^b21f (mean)

CI 21r (mean)

HDACi plus erlotinib

HDACi (24 h), washout then

erlotinib (6 days)

0.32

0.28

H520

DU-145^a

MDA-MB-231

NSCLC

prostate carcinoma

breast carcinoma

0.54

0.63

0.94

0.85

0.61

0.773

HDACi plus decitabine

HDACi plus tosedostat

decitabine (48 h)

then decitabine plus HDACi (72 h)

MDA-MB-435

BxPC-3

breast ductal carcinoma

pancreatic carcinoma

0.74

ND^c

1.097

0.262

HDACi (24 h), washout then

CHR-2797 (72 h)

RPMI-7951

H23

malignant melanoma

NSCLC

ND^c

0.288

0.780

^a5 day erlotinib for DU-145. ^bCI values indicate the degree of synergy according to the following system. CI: >1.1, antagonism; 0.90-1.10, nearly

additive; 0.70-0.90, slight to moderate synergism; 0.30-0.70, synergism; 0.10-0.30, strong synergism; <0.10, very strong synergism. ^cNot determined.

Table 9. Cell Cycle Effects of 21f and 21r^a

mean ( SD, n = 6 replicates. Results are summarized as GI₅₀

values, i.e., the concentration of inhibitor that inhibits 50% of

the vehicle response.

HCT116 cells after 15 h of drug treatment (%)

compd

sub-G1

G1

S

G2/M

Western Blotting and Flow Cytometry. HCT116 cells were

seeded at 2 ꢀ 10⁴cells/mL and 24 h later treated with the stated

doses of compound or vehicle for 6, 15, or 30 h. Samples were

harvested on ice and washed twice with ice cold PBS before (a)

lysing the pellet in RIPA buffer to prepare SDS-PAGE samples or

(b) fixing pellet overnight at 1 ꢀ 10⁶cells/mL in 70% ethanol/30%

PBS (ice cold) before preparing samples for FACS. Gel samples

were loaded at 10 μg/lane on 4-12% acrylamide gels, transferred to

nitrocellulose, and stained with primary antibodies to acetylated

R-tubulin, acetylated H3 lysine 9 (K9), GAPDH, and PARP. Signal

was visualized using ECL and film. FACS samples were prepared

by washing twice with PBS before resuspending cells gently in

propidium iodide/RNase solution for 30 min before reading the

output using a FACScanto. Cells were gated to give populations of

sub-G1, G1, S, and G2/M phases. Numbers shown represent the

percent of each population from the total number of single cells

counted (10 000 per sample).

vehicle treated

21f

5

23

21

19

36

33

21

35

21

11

10

14

38

33

48

32

21r

SAHA

^aHuman HCT116 colon carcinoma cells were incubated with 300 nM

inhibitor. DNA content of nuclei was evaluated using propidium iodide

staining followed by flow cytometry analysis, and the number of cells in

sub-G1, G1, S, and G2/M phases was calculated as a percentage of

control. Results are expressed as percent of total cells, and a representa-

tive experiment out of three is shown.

137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, pH 8.0). Com-

pounds (30 μM in 0.3% DMSO) were preincubated for 5 min

with the diluted enzyme (10 μg of HeLa cell nuclear extract

protein) at room temperature in a 15 μL volume. The reaction

was initiated by the addition of the substrate (15 μL, 0.125 mM).

After 15 min of incubation at room temperature the developer

concentrate (25 μL), which contained 2 μM trichostatin A to

stop the reaction, was added and the plate shaken. After the

mixture was left to stand for 10 min, the fluorescence signal was

read on a Victor II plate reader with excitation at 355 nm and

emission at 455 nm. On each assay plate, column 1 contained

DMSO alone for the totals and column 12 had buffer added

rather than enzyme to generate the blank values. For the IC₅₀

determinations, each compound was assayed at eight concen-

trations in duplicate and the data were analyzed in Graph Pad

Prism. Preliminary kinetic assays with the Fluor de Lys sub-

strate produced a K_mof 60 ( 10 μM (Biomol K_m= 54 μM), so

all assays were run at a substrate concentration of 62.5 μM. Under

these conditions, enzyme activity was linear up to 15 min of

incubation and 40 μg of protein. Measurement of inhibitor activity

for HDAC isoforms was performed by BPS Biosciences (San Diego,

CA) using human recombinant enzymes in a fluorogenic assay kit.

Protocols are available at http://www.bpsbioscience.com.

Cellular Proliferation Assays. Cells were seeded in 96-well

BD-Falcon plates (Becton Dickinson) at a density of (1-5) ꢀ

10³cells per well in the appropriate serum-containing culture

medium and cultured at 37 °C in a humidified 5% (v/v) CO₂

incubator for 24 h. Samples (50 μL) of different HDAC inhibitor

concentrations (0.6-10000 nM), diluted in the relevant culture

medium, were transferred to the wells containing the cells for a

further 72 h. During the final 4 h of the incubation, cells were

pulsed with 10 μL of the tetrazolium salt Cell Proliferation

Reagent, WST-1 (Roche). In the presence of viable cells WST-1

is metabolized to formazan product which is measured spectro-

photometrically at 450 nm against a background media control

(reference wavelength of 650 nm). Antiproliferative effects of

HDAC inhibitors on the cells were expressed as percentage

inhibition of the vehicle response and were the plotted as the

Thymidine Incorporation Proliferation Assays for Synergy

Studies. Cells were seeded in 96-well BD-Falcon plates (Becton

Dickinson, Oxford, U.K.) at a density of (1-5) ꢀ 10³cells per well

in the appropriate serum containing culture medium and cultured

at 37 °C in a humidified 5% CO₂incubator for 24 h. Compounds

to be tested were serially diluted in the relevant culture medium and

then added to the wells for a further 72 h before pulsing the cells for

4 h with 0.4 μCi per well of ³H-methylthymidine (specific activity

1 mCi/mL Amersham Biosciences, U.K.). For synergy experiments,

minor dosing modifications were carried out to take account of the

necessary scheduling differences required when studying more

than one drug combination. Cells were then harvested onto glass

fiber filter mats (GF/C Perkin-Elmer LAS, U.K.) using a Tomtec

harvester and then counted on a 1450 MicroBeta scintillation

counter to determine the amount of radioactive thymidine incor-

porated into cellular DNA. Data were expressed as a mean

percentage ( SD, relative to vehicle response, which measures

3

the amount of H-thymidine incorporation into cells in the pre-

sence of medium containing 0.1% DMSO and no test compound.

Results are calculated as the GI₅₀or concentration of compound

that inhibited the vehicle response by 50%.

In Vitro ADME Studies. Microsomal stability studies were

performed using published methods.³³Inhibition studies for

CYP2D6 and 3A4 were performed using previously described

methods.³⁴IC₅₀values for CYP2C19 were determined by

Cyprotex (U.K.).

Human Tumour Xenograft Studies. Female CrTac:NCr-Fox1-

(nu) athymic mice 6-8 weeks of age bred in-house at ICR were

inoculated bilaterally sc with 2 million human HCT116 colon

carcinoma cells. Daily treatment with 21f (50 mg/kg po) com-

menced on day 6, when tumors were established (approximately

5 mm mean diameter). Controls received an equal volume of

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8671

Figure 8. (a) Efficacy of 21f 50 mg/kg po daily and (b) histone acetylation in HCT116 tumor tissue from mice treated with 21f. (a) Relative

tumor volumes in HCT116 human tumor mouse xenograft study. Compound 21f (50 mg/kg po in 10% DMSO-saline) was administered for 16

days to female CrTac:NCr-Fox1(nu) mice bearing established HCT116 colon carcinoma xenografts. (b) Histone acetylation in HCT116 tumor

tissue treated with 21f as in (a). Tumour tissue was obtained, and lysates were prepared 0.5, 1.0, and 4 h following the last dose of 21f. The

samples were analyzed using a quantitative sandwich ELISA with electrochemiluminescent end point (see Supporting Information). Histone

acetylation was also measured by Western blot using an antibody to acetylated histone H3 (Abcam) and GAPDH as loading control (see

Supporting Information). Gray bars are 21f-treated samples. Black bars show densitometry results from the Western blot normalized to

GAPDH, and white bars represent the samples from vehicle control animals. C = samples from vehicle treated mice.

Figure 9. (a) Relative tumor volumes in HCT116 tumor xenografts after treatment with 21f and 21r. Compound 21f (75 mg/kg po) and 21r (50

mg/kg po) were administered for 14 days to female nude BALB/c mice in 5% DMSO-saline bearing the HCT116 colon carcinoma xenograft

(EpiStem Ltd.). (b) Plasma and tumor concentrations of 21f and 21r.

Chemistry General Methods. All nonaqueous reactions were

carried out under a nitrogen atmosphere unless otherwise noted.

All solvents employed were commercially available HPLC

1

grade. H NMR spectra were determined with a Bruker AV

spectrometer at 300 MHz. Chemical shifts are reported in parts

per million (δ) relative to residual chloroform (7.26 ppm) or

dimethylsulfoxide (2.54 ppm) as internal reference with cou-

pling constants (J) reported in hertz (Hz). The peak shapes are

reported as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,

multiplet; br, broad. Analytical HPLC/MS was performed on

an Agilent HP1100 LC system using reverse phase Luna C18

columns (3 μm, 50 mm ꢀ 4.6 mm), gradient 5-95% B (A =

water/0.1% formic acid, B = acetonitrile/0.1% formic acid)

over 2.25 min, flow rate of 2.25 mL/min. UV spectra were

recorded at 220 and 254 nm using a G1315B DAD detector.

Mass spectra were obtained over the range m/z 150-800 on a

Figure 10. Growth curves of LoVo human colon xenografts after

LC/MSD SL G1956B detector. Data were integrated and

reported using ChemStation and ChemStation Data Browser

software. The purity of all the above-mentioned compounds was

found to be above 95%. Reverse phase HPLC purifications were

performed on Gilson preparative systems using reverse phase

Axia prep Luna C18 columns (10 μm, 100 mm ꢀ 21.2 mm),

gradient 0-100% B (A = water/0.05% TFA, B = acetonitrile/

0.05% TFA) over 10 min, flow rate of 25 mL/min, monitored by

UV detection at 220 or 254 nm. Thin-layer chromatography

(TLC) analysis was performed with Kieselgel 60 F₂₅₄(Merck)

plates and visualized using UV light. The terms “concentrated”

and “evaporated” refer to removal of solvents using a rotary

treatment with 21r. Relative tumor volumes in LoVo tumor xeno-

graft study. Compound 21r (25 and 50 mg/kg po) was administered

for 19 days to female nude BALB/c mice in 5% DMSO-saline

bearing the LoVo colon carcinoma xenograft (EpiStem Ltd.).

vehicle alone, 10% DMSO in saline). Tumor measurements and

body weights were recorded three times weekly. The study was

terminated on day 16, and plasma, spleen, and tumor samples

were obtained and stored frozen for later PK and biomarker

assays. Additional efficacy studies in HCT116 and LoVo human

tumor xenograft bearing mice using 21r were carried out at

Epistem Ltd. (U.K.).

8672 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

evaporator at water aspirator pressure with a bath temperature

equal to or less than 40 °C. Unless otherwise noted, reagents

were obtained from commercial sources and were used without

further purification.

(1.2 equiv) and HOBt (1.2 equiv) in DMF at room temperature

under a nitrogen atmosphere. O-(1-Isobutoxyethyl)hydroxylamine

7 (5 equiv) was then added followed by triethylamine (5 equiv) and

the mixture left to stir for 16 h. The mixture was then diluted with

H₂O and extracted with CH₂Cl₂. The combined organic layers were

dried over MgSO₄, and solvent was removed in vacuo. The residue

was purified by column chromatography using an eluent of 0-10%

MeOH in CH₂Cl₂to give the product. The protected hydroxamate

product (1 equiv) was stirred in CH₂Cl₂at room temperature

and 4 M HCl in dioxane (2 equiv) added. The mixture was

stirred for 10 min and then the solvent removed in vacuo. The

residue was purified by preparative HPLC to give the product as a

TFA salt.

Ethyl 2-(Methylsulfonyl)pyrimidine-5-carboxylate 2: Step 1.

To a suspension of ethyl 4-chloro-2-(methylthio)pyrimidine-

5-carboxylate 1 (5.0 g, 21.5 mmol) in THF (25 mL) at 85 °C

was added slowly dropwise powdered zinc (7.0 g, 107.7 mmol) in

water (25 mL). Following complete dissolution of the zinc

powder, tert-BuOH (1 mL) was added and heating was con-

tinued at 90 °C with vigorous stirring for 18 h. The mixture was

cooled to room temperature and filtered through a Celite pad

which was washed with further EtOAc (20 mL). The combined

filtrates were concentrated, redissolved in EtOAc (30 mL), and

washed with water. The EtOAc layer was dried (Na₂SO₄),

filtered, and concentrated to dryness and the residue purified

by flash column chromatography [eluting with 0-100% EtOAc in

hexane] to give ethyl 2-(methylthio)pyrimidine-5-carboxylate as a

colorless oil (2.45 g, 57%). m/z 199 [M þ H]^þ. ¹H NMR (300 MHz,

DMSO-d₆) δ: 9.0 (2H, s), 4.35 (2H, q, J=7.2Hz), 2.60(3H, s), 1.30

(3H, t, J =7.2 Hz).

Step 2. To a stirred solution of ethyl 2-(methylthio)pyrimidine-

5-carboxylate (2.9 g, 14.64 mmol) in dry THF (76 mL) was added

77% mCPBA (11.0 g, 64.4 mmol) at 0 °C under N₂. The reaction

mixture was allowed to warm to room temperature, stirred for 2 h

before evaporating to dryness. The residue was purified by flash

column chromatography [eluting with, 100% hexane followed by

1:5:3 CH₂Cl₂/heptane/Et₂O, followed by 1:1:1 CH₂Cl₂/heptane/

Et₂O] to give compound 2 as a white solid (10.0 g, 66%). m/z 231

[M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.50 (2H, s), 4.40

(2H, q, J = 7.2 Hz), 3.50 (3H, s), 1.40 (3H, t, J = 7.2 Hz).

tert-Butyl 6-Aminoazabicyclo[3.1.0]hexane-3-carboxylate 8. Step 1.

Preparation of 3-Benzyl-6-nitro-3-azabicyclo[3.1.0]hexane-2,4-

dione 4. To a solution of N-benzylmaleimide (5.0 g, 26.7 mmol) in

CH₃CN (334 mL) was added bromonitromethane (1.87 mL, 26.7

mmol). K₂CO₃(3.69 g, 26.7 mmol) was added and the reaction

mixture was vigorously stirred at room temperature. After 4 h an

additional portion of bromonitromethane (0.2 mL, 2.8 mmol) was

added. Further bromonitromethane portions (0.2 mL, 2.8 mmol)

were added at further 4 h intervals (x4). After 48 h the reaction

mixture was evaporated to dryness and the residue was purified by

column chromatography [eluting with, 100% CH₂Cl₂], giving 4 as

a white solid (3.0 g, 42%). ¹H NMR (300 MHz, CDCl₃) δ:

7.35-7.24 (5H, m), 4.52 (2H, s), 4.41 (1H, s,), 3.33 (2H, s).

Step 2. Preparation of 3-Benzyl-6-nitro-3-azabicyclo[3.1.0]hexane

5. To a solution of 3-benzyl-6-nitro-3-azabicyclo[3.1.0]hexane-2,4-

dione 4 (3.0 g, 12.2 mmol) in dry THF (30 mL) was added NaBH₄

(1.15 g, 30.48 mmol). The mixture was stirred for 15 min at room

Modeling and Calculated Properties. Compounds were docked

with Glide, version 55211 (Schrodinger Inc., Portland, OR, U.S.)

using the SP settings. The protein structure used was PDB code

1T67, which was prepared in MOE, version 2008.1001 (Chemical

Computing Group, Montreal, Canada). Initially the software

perceived the ligand as having a double bond between the N and

O atoms of the hydroxamic acid. This was manually adjusted to a

single bond, and the oxygen was assigned a formal negative charge.

The Protonate 3D tool was then used to assign the remaining

ionization states, add hydrogen atoms, and adjust tautomers and

conformers of His, Asn, and Gln residues. This structure was then

transferred to Maestro (Schrodinger Inc., Portland, OR, U.S.)

where all waters were removed except Wat392 (the 11th one as read

from the PDB file). This water appears to play an important role in

the ligand binding by bridging between its amide oxygen and

His180. The positions of the hydrogen atoms were then refined

using the OPLS2005 force field in Maestro’s protein preparation

wizard before generating a Glide grid for docking purposes. This

included a constraint to ensure that the ligands bind to the zinc ion

of the active site.

2

˚

PSA values (A ) were calculated using Pipeline Pilot, version

7.01.100 (Accelrys, Cambridge, U.K.). pK_avalues were calcu-

lated using Structure Designer (ACD Labs, Toronto, Canada).

Method A. Reductive Amination Reactions of Compound (8).

Compound 8 (1.2 equiv) was stirred in MeOH at room tempera-

ture under N₂, and aldehyde (1 equiv) was added. The resultant

solution was stirred for 3 h. After thistime, NaBH₄(1.7 equiv) was

added and the solution stirred for 10 min. Then 1 M NaOH (3-4

equiv) was added, forming an opaque white solution that was

stirred for 20 min. Water was then added and the solution

extracted with Et₂O. The combined organic extracts were dried

(MgSO₄) and solvent was removed in vacuo to give the product

which was either used without purification or purified by flash

column chromatography.

Method B. Boc Deprotection of N-Substituted tert-Butyl 6-Amino-

3-azabicyclo[3,1,0]hexane-3-carboxylates. A solution of the Boc-

protected 6-aminoazabicyclo[3,1,0]hexane(1 equiv) was stirredin

4 M HCl in dioxane (8 equiv) under N₂at room temperature

for 0.5 h. The solvent was removed under reduced pressure to give

the product amine hydrochloride salt which was used without

purification.

Method C. Reaction of N-Substituted 6-Amino-3-azabicyclo-

[3,1,0]hexanes with Ethyl 2-(Methylthio)pyrimidine-5-carboxylate

and Hydrolysis of the Resulting Ethyl 2-{6-Amino]-3-azabicyclo-

[3.1.0]hex-3-yl}pyrimidine-5-carboxylates. K₂CO₃(3 equiv) was

added to a stirred suspension of the N-substituted 6-amino-

3-azabicyclo[3.1.0]hexane trifluoroacetate (1 equiv) in CH₃CN

at room temperature under N₂. A solution of compound 2

(1 equiv) in CH₃CN was added dropwise over 5 min, leading to

the formation of a precipitate which was stirred at room tempera-

ture overnight. The reaction mixture was diluted with EtOAc,

giving a suspension that was washed with water. The precipitate in

the organic phase was isolated by filtration, washed with TBME,

and air-dried to give the product. The product may be used without

purification or purified by flash column chromatography. Aque-

ous 1 M NaOH (10 equiv) was added to a solution of the resulting

product (1 equiv) in THF and MeOH at room temperature. The

reaction mixture was stirred at room temperature for 18 h. The

organic solvents were removed in vacuo, and the resultant aqueous

solution was acidified to pH ∼5 with 1 M aq HCl. The precipitate

was isolated by filtration, washed with H₂O, and dried by azeo-

troping with toluene, giving the product which was used without

purification or purified by trituration.

temperature under N₂. BF₃THF complex (3.15 mL, 13.4 mmol)

3

was added dropwise and the mixture heated at 40 °C for 4 h.

Further NaBH₄(0.15 g, 3.96 mmol) was added followed by

BF₃THF complex (0.32 mL, 1.4 mmol). Heating was continued

3

at 45 °C for 30 min. A mixture of THF/H₂O (1:1 v/v, 60 mL) was

added dropwise with stirring. The resulting mixture was heated at

50 °C for 1 h before standing at room temperature for 16 h. The

THF was removed under reduced pressure and the resulting

aqueous mixture was extracted with EtOAc (3 ꢀ 50 mL), dried

(MgSO₄) and evaporated to dryness to give 5 as a yellow oil (2.47 g,

93%). m/z 219 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ: 7.63

(2H, br m), 7.44-7.42 (3H, m), 5.38 (1H, br s), 4.33 (2H, br s), 3.57

(4H, br s), 2.91 (24H, s).

Method D. Hydroxamate Formation Using O-(1-Isobutoxyethyl)-

hydroxylamine (7) Followed by Deprotection. The pyrimidine-

5-carboxylate (1 equiv) was stirred with EDC hydrochloride

Article

Step 3. Preparation of 6-Nitro-3-azabicyclo[3.1.0]hexane Hydro-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8673

concentrated in vacuo and then CH₂Cl₂(100 mL) was added and

re-evaporated in vacuo three times to remove excess TFA, giving

13a as the trifluoroacetate salt (crude yield 34.0 g). This product

was used in the next step without purification. ¹H NMR (400 MHz,

DMSO-d₆) δ: 9.20 (1H, br s), 8.48 (1H, s), 8.33 (1H, br s), 8.23-8.18

(3H, m), 8.08 (1H, d, J=8.7 Hz), 7.83 (1H, d, J=7.9 Hz), 7.75-7.70

(2H, m), 3.22 (4H, m), 2.20 (1H, m), 1.86 (2H, s).

chloride 6. To a solution of 3-benzyl-6-nitro-3-azabicyclo[3.1.0]-

hexane 5 (2.47 g, 11.3 mmol) in 1,2-dichloroethane (5 mL) was

added 1-chloroethylchloroformate (1.83 mL, 16.9 mmol) at 0 °C.

The reaction mixture was heated to 55 °C for 4 h. Further

1-chloroethylchloroformate (0.5 mL) was added, and heating

continued for 2 h. The reaction mixture was then evaporated to

dryness. MeOH (15 mL) was added, and the reaction mixture was

heated at 65 °C for 3 h. Concentrated HCl (1 mL) was added and

the mixture stirred at room temperature for 2 h. A precipitate

formed which was isolated by filtration and washed with Et₂O to

Step 2. Preparation of 2-{6-[(2-Naphthylsulfonyl)amino]-3-

azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxylic Acid 14a. Follow-

ing method C, K₂CO₃(28.33 g, 205.29 mmol) was added to a stirred

suspension of 6-[(2-naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]-

hexane trifluoroacetate 13a (27.50 g, 68.43 mmol) in CH₃CN

(300 mL) at room temperature under N₂. A solution of compound

2 (15.74 g, 68.43 mmol) in CH₃CN (50 mL) was added dropwise

over 5 min, leading to the formation of a precipitate which was

stirred at room temperature overnight. The reaction mixture was

diluted with EtOAc (250 mL), giving a suspension which was

washed with water (2 ꢀ 250 mL). The precipitate in the organic

phase was isolated by filtration, washed with TBME, and air-dried

to give ethyl 2-{6-[(2-naphthylsulfonyl)amino]-3-azabicyclo-

[3.1.0]hex-3-yl}pyrimidine-5-carboxylate as a white solid (25.40 g,

85%). m/z 439 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ: 8.70

(2H, s), 8.50 (1H, m), 8.22-8.15 (3H, m), 8.04 (1H, d, J =

8.3 Hz), 7.84 (1H, m), 7.67-7.71 (2H, m), 4.23 (2H, q, J = 7.3

Hz)), 3.70-3.67 (2H, m), 3.53-3.50 (2H, m), 1.90 (1H, m), 1.82

(2H, s), 1.26 (3H, t, J = 7.3 Hz). Aqueous 1 M NaOH (500 mL)

was added to a solution of ethyl 2-{6-[(2-naphthylsulfonyl)amino]-

3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxylate (25.40 g,

58.00 mmol) in THF (500 mL) and MeOH (100 mL) at room

temperature, and the reaction mixture was stirred for 18 h. The

organic solvents were removed in vacuo, and the resultant aqueous

solution was acidified to pH 5 with 1 M aqueous HCl. A heavy

white precipitate was formed which was isolated by filtration,

washed with H₂O, and dried by azeotroping with toluene to give

2-{6-[(2-naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxylic acid 14a as a white solid (22.19 g, 93%). m/z

411 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ:8.83(2H, s), 8.66

(1H, s), 8.36-8.31 (3H, m), 8.21 (1H, d, J = 8.7 Hz), 8.01 (1H, m),

7.88-7.84 (2H, m), 3.89-3.82 (2H, m), 3.67-3.64 (2H, m), 2.05

(1H, s), 1.97 (2H, s).

1

give 6 as a gray powder (464 mg, 25%). H NMR (300 MHz,

DMSO-d₆) δ: 9.50 (2H, br s), 4.75 (1H, s), 3.60-3.31 (4H, m),

2.90 (2H, s).

Step 4. Preparation of tert-Butyl 6-Nitro-3-azabicyclo[3.1.0]-

hexane-3-carboxylate 7. 6-Nitro-3-azabicyclo[3.1.0]hexane hydro-

chloride 6 (464 mg, 2.82 mmol) was suspended in dry CH₂Cl₂

(10 mL) and cooled to 0 °C. Di-tert-butyl dicarbonate (677 mg, 3.10

mmol) was added followed by DMAP (1 crystal) and triethylamine

(0.43 mL, 3.10 mmol). The reaction mixture was stirred at room

temperature for 5 h. The solvent was removed under reduced

pressure to give a white solid. Purification by flash column chro-

matography [eluting with 25% EtOAc in heptane) gave 7 as a white

solid (592 mg, 92%). ¹H NMR (300 MHz, CDCl₃) δ: 4.10 (1H, t),

3.77 (2H, br m), 3.51 (2H, br d), 2.67 (2H, s), 1.46 (9H, s).

Step 5. tert-Butyl 6-Aminoazabicyclo[3.1.0]hexane-3-carbox-

ylate 8. tert-Butyl 6-nitro-3-azabicyclo[3.1.0]hexane-3-carbox-

ylate 7 (592 mg, 2.59 mmol) was reduced in the presence of 10%

Pd/C (20 mg) in EtOH(5 mL) under H₂(balloonpressure) for18h

at room temperature. The catalyst was removed by filtration

through Celite washing with EtOH and the filtrate concentrated

to give 8 as a pale yellow oil (487 mg, 95%). ¹H NMR (300 MHz,

DMSO-d₆) δ: 3.54 (1H, d, J = 10.6 Hz), 3.51 (1H, d, J = 10.7

Hz), 3.38-3.32 (2H, br m), 2.12 (1H, t, J = 0.8 Hz), 2.01-1.93

(2H,br m), 1.49 (2H, t, J = 10.5 Hz), 1.44 (9H, s).

6-Fluoroquinoline-2-carbaldehyde 10. 6-Fluoro-2-methylqui-

noline (1.0 g, 6.2 mmol) was stirred in dioxane (50 mL) at room

temperature under a nitrogen atmosphere. SeO₂(0.86 g, 7.4

mmol) was added and the mixture heated to 100 °C for 1 h,

during which time the mixture changed from an opaque brown

color to a clear dark red solution. The mixture was cooled to

room temperature, filtered and the filtrate concentrated in

vacuo. The residue was purified by column chromatography

[eluting with 0-3% MeOH in CH₂Cl₂] to give the product as an

off-white solid (944 mg, 87%). m/z 239 [M þ H]^þ. ¹H NMR (300

MHz, DMSO-d₆) δ: 10.11 (1H, s), 8.60 (1H, d, J = 11.0 Hz),

8.32 (1H, dd, J = 7.6, 11.0 Hz), 8.04 (1H, d, J = 11.0 Hz), 7.96

(1H, dd, J = 4.4, 11.0 Hz), 7.85 (1H, td, J = 4.4, 11.0 Hz).

N-Hydroxy-2-{6-[(2-naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]-

hex-3-yl}pyrimidine-5-carboxamide Trifluoroacetate 15a: Step 1.

6-[(2-Naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hexane Trifluor-

oacetate 13a. 2-Naphthalenesulfonyl chloride (20.07 g, 97.39 mmol)

was added to a solution of tert-butyl 6-aminoazabicyclo-

[3.1.0]hexane-3-carboxylate 5 (1.69 g, 8.54 mmol) and triethyla-

mine (24.7 mL, 177.07 mmol) in anhydrous CH₂Cl₂(270 mL) at

0 °C under N₂, giving a light brown solution which was allowed to

warm to room temperature overnight. The reaction mixture was

diluted with CH₂Cl₂(200 mL) and saturated NaHCO₃(200 mL).

The organic phase was separated and washed with H₂O(2ꢀ 200 mL),

dried (Na₂SO₄), filtered, and concentrated in vacuo to give a pale

yellow oil. Trituration with 5% TBME in heptane (50 mL) gave a

solid which was isolated by filtration and washed with heptane to

afford 13a as a white solid (27.99 g, 82%). m/z 389 [M þ H]^þ. ¹H

NMR (300 MHz, CDCl₃) δ: 8.47 (1H, m), 8.02-7.99 (2H, m),

7.93 (1H, d, J = 7.9 Hz), 7.85 (1H, m), 7.68-7.62 (2H, m), 5.12

(1H, br s), 3.56-3.41 (2H, m), 3.34-3.29 (2H, m), 1.96 (1H, m),

1.82 (2H, m), 1.35 (9H, s). A solution of tert-butyl 6-[(2-

naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hexane-3-carboxylate

(27.50 g, 68.43 mmol) in 20% TFA in CH₂Cl₂(300 mL) was

stirred at room temperature for 2 h. The reaction mixture was

Step 3. Preparation of N-Hydroxy-2-[6-(naphthalene-2-sulfonyl-

amino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxamide 15a.

FollowingmethodD, EDChydrochloride(7.27g, 37.9mmol)was

added to a suspension of 2-{6-[(2-naphthylsulfonyl)amino]-3-

azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxylic acid 14a (22.19 g,

54.12 mmol) in anhydrous CH₂Cl₂(100 mL) and anhydrous THF

(500 mL) at room temperature under N₂. Triethylamine (22.6 mL,

162.14 mmol) was added followed by HOBt (8.78 g, 64.97 mmol) and

O-(1-isobutoxyethyl)hydroxylamine 7 (8.9 mL, 64.89 mmol). After

being stirred at room temperature for 6 days, the reaction mixture

was re-treated with EDC hydrochloride (10.25 g, 53.47 mmol), HOBt

(7.22 g, 53.43 mmol), and triethylamine (19.4 mL, 139.0 mmol). After

being stirred for another 3 days at room temperature, the reaction

mixture was evaporated to dryness and suspended in EtOAc

(100 mL) and water (100 mL). A white solid was collected by

filtration, washed with EtOAc (50 mL), water (50 mL), MeOH

(50 mL), and dried in vacuo to give a white solid. This was stirred in

EtOH (1500 mL) at 60 °C for 1 h, giving a white suspension which

was cooled to room temperature, filtered, and air-dried to give N-(1-

isobutoxyethoxy)-2-[6-(naphthalene-2-sulfonylamino)-3-azabicyclo-

[3.1.0]hex-3-yl]pyrimidine-5-carboxamide 15a as a white solid (24.0 g,

87%). m/z511 [M þ H]^þ. A solution of 4 M HCl in dioxane (350 mL,

1.4 mol) was added in portions over 5 min to N-(1-isobutoxyethoxy)-

2-[6-(naphthalene-2-sulfonylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyri-

midine-5-carboxamide (10.0 g, 19.65 mmol) at room temperature

with vigorous stirring under N₂. After 2 h CH₂Cl₂(20 mL) was

added followedby another portion of 4 M HCl indioxane (20 mL)

and stirring was continued for a further 2.5 h. The reaction

mixture was evaporated in vacuo to ∼200 mL volume, and

CH₂Cl₂(200 mL) was added in portions effecting precipitation.

8674 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

The resultant white precipitate was isolated by filtration and

washed with CH₂Cl₂(5 mL), giving 15a as a white powder (6.25

g, 75%). m/z 426 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ:

11.02 (1H, br s), 8.97 (1H, br s), 8.58 (2H, s), 8.50 (1H, s),

8.24-8.10 (3H, m), 8.05 (1H, d, J = 8.8 Hz), 7.83 (1H, dd, J =

1.8, 8.8 Hz), 7.80-7.65 (2H, m), 3.65 (2H, d, J = 12.0 Hz), 3.45

(2H, d, J = 12.0 Hz), 3.20 (1H, s), 1.80 (1H, s). Anal. Calcd for

C₂₁H₂₆ClN₅O₆S: C, 49.27; H, 5.12; Cl, 6.92; N, 13.68. Found: C,

49.70; H, 5.09; Cl, 7.10; N, 13.55.

as a yellow oil which was used in the next step without purification.

m/z 389 [M þ H]^þ. Ethyl 2-[6-(naphth-2-ylmethyl)-3-azabicyclo-

[3.1.0]hex-3-yl]pyrimidine-5-carboxylate (0.96 mmol) was hydro-

lyzed with 1 M NaOH (10 mL, 10 mmol) in THF (10 mL) to give

2-[6-(naphth-2-ylmethyl)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-

5-carboxylic acid 20a (70 mg, 19% over 4 steps) as a colorless solid

which was used in the subsequent step without purification. m/z

361 [M þ H]^þ. Following method D, 2-{6-[(2-naphth-2-ylmethyl)-

amino]-3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxylic acid

(70 mg, 0.194 mmol) was stirred with EDC hydrochloride

(56 mg, 0.29 mmol) and HOBt (39 mg, 0.29 mmol), triethylamine

(135 μL, 0.97 mmol), and O-(1-isobutoxyethyl)hydroxylamine

(270 μL, 1.94 mmol). In this case after quenching the reaction

with water (10 mL) the layers were separated and the aqueous

layer was re-extracted with CH₂Cl₂(3 ꢀ 10 mL). The combined

organic layers were washed with 0.5 M HCl and the organic layer

was dried (MgSO₄) and evaporated in vacuo to give N-hydroxy-

2-{6-[(2-naphthylmethyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxamide 21a (15 mg, 20%). m/z 376 [M þ H]^þ. ¹H

NMR (300 MHz, DMSO-d₆) δ: 11.10 (1H, br s), 9.61 (1H, br s),

9.01 (1H, br s), 8.66 (2H, s), 8.07 (1H, s), 7.97 (3H, m), 7.67 (1H,

dd, J = 8.4, 1.8 Hz), 7.57 (2H, m), 4.41 (2H, s), 3.80 (2H, d, J =

11.7 Hz), 3.56 (2H, d, J = 11.7 Hz), 2.57 (1H, m), 2.22 (2H, br s).

2-{6-[(4-Chlorobenzyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}-

N-hydroxypyrimidine-5-carboxamide Bistrifluoroacetate 21f.

Following method A, tert-butyl 6-aminoazabicyclo[3.1.0]hexane-3-

carboxylate 8 (200 mg, 1.01 mmol), 4-chlorobenzaldehyde (135 mg,

0.96 mmol), and sodium borohydride (36 mg, 0.96 mmol) gave tert-

butyl 6-(4-chlorobenzylamino)-3-azabicyclo[3.1.0]hexane-3-car-

boxylate which was used in the subsequent step without purifica-

tion. m/z 323/325 [M þ H]^þ. Following method B, tert-butyl 6-(4-

chlorobenzylamino)-3-azabicyclo[3.1.0]hexane-3-carboxylate

(0.96 mmol) was stirred in 4 M HCl in dioxane (2 mL) at room

temperature for 0.5 h to give N-(4-chlorobenzyl)-3-azabicyclo-

[3.1.0]hexan-6-amine hydrochloride 19f which was used in the

next step without purification. m/z 223/225 [M þ H]^þ. Following

method C, N-6-(4-chlorobenzyl)-3-azabicyclo[3.1.0]hexan-6-amine

hydrochloride 19f (0.96 mmol), ethyl 2-(methylsulfonyl)pyrimidine-

5-carboxylate 2 (221 mg, 0.96 mmol), and potassium carbonate

(1.32 g, 9.6 mmol) gave ethyl 2-{6-[(4-chlorobenzyl)amino]-3-

azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxylate as a yellow oil

which was used in the next step without purification. m/z 373/375

[M þ H]^þ. Ethyl 2-{6-[(4-chlorobenzyl)amino]-3-azabicyclo[3.1.0]-

hex-3-yl}pyrimidine-5-carboxylate (0.96 mmol) was hydrolyzed

with 1 M NaOH (10 mL, 10 mmol) in THF (10 mL) to give

2-{6-[(4-chlorobenzyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxylic acid 20f (166 mg, 50% over four steps) as a

colorless solid which was used in the subsequent step without

purification. m/z 345/347 [M þ H]^þ. Following method D,

2-{6-[(4-chlorobenzyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxylic acid 20f (122 mg, 0.14 mmol), EDC hydro-

chloride (121 mg, 0.63 mmol), HOBt (81 mg, 0.63 mmol), O-(1-

isobutoxyethyl)hydroxylamine (373 μL, 2.62 mmol), and triethy-

lamine (365 μL, 2.62 mmol) gave 2-{6-[(4-chlorobenzyl)amino]-

3-azabicyclo[3.1.0]hex-3-yl}-N-(1-isobutoxyethoxy)pyrimidine-5-

carboxamide (128 mg, 58%) after purification by flash column

chromatography [eluting with 0-10% MeOH/CH₂Cl₂]. m/z 460/

462 [M þ H]^þ. 2-{6-[(4-chlorobenzyl)amino]-3-azabicyclo[3.1.0]-

hex-3-yl}-N-(1-isobutoxyethoxy)pyrimidine-5-carboxamide (128 mg,

0.28 mmol) was stirred with TFA (0.5 mL) in 1:1 v/v CH₂Cl₂/

MeOH (4 mL) overnight. The solvent was removed in vacuo and

the residue purified by preparative HPLC to give 2-{6-[(4-chloro-

benzyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}-N-hydroxypyrimidine-

5-carboxamide bistrifluoroacetate 21f (41 mg, 25%) as a colorless

solid. m/z 360/362 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ:

11.07 (1H, br s), 9.26 (1H, br s), 9.04 (1H, br s), 8.67 (2H, s), 7.54

(4H, m), 4.27 (2H, s), 3.71 (2H, d, J = 11.7 Hz), 3.57 (2H, d, J =

11.7 Hz) 2.57 (1H, br s), 2.13 (2H, br s).

N-Hydroxy-2-{6-[(naphthalene-2-carbonyl)amino]-3-azabicyclo-

[3.1.0]hex-3-yl}-pyrimidine-5-carboxamide 18a. To a stirred solu-

tion of tert-butyl 6-aminoazabicyclo[3.1.0]hexane-3-carboxylate 8

(300 mg, 1.51 mmol) in pyridine (1 mL) at 0 °C was added

2-naphthoyl chloride (316 mg, 1.66 mmol). The mixture was stirred

for 1 h, and water (8 mL) was then added. The resulting precipitate

was collected by filtration and dried under vacuum to afford tert-

butyl 6-(2-naphthoylamino)-3-azabicyclo[3.1.0]hexane-3-carbox-

1

ylate (406 mg, 76%). H NMR (300 MHz, CDCl₃) δ: 8.26 (1H,

s), 7.88-7.95 (3H, m), 7.81 (1H, d, J = 1.8, 8.8 Hz), 7.60-7.54

(2H, m), 6.37 (1H, br s), 3.82 (2H, d, J = 11.4 Hz), 3.50-3.40 (2H,

m), 2.71 (1H, d), 1.85 (2H, s), 1.47 (9H, s). Following method B,

tert-butyl 6-(2-naphthoylamino)-3-azabicyclo[3.1.0]hexane-3-car-

boxylate (406 mg, 1.15 mmol) was stirred in 4 M HCl in dioxane

(10 mL) to give N-(3-azabicyclo[3.1.0]hex-6-yl)naphthalene-2-car-

boxamide hydrochloride 16a as a pink solid which was carried

forward without purification. m/z 353 [M þ H]^þ. Following

method C, compound 16a (1.15 mmol), compound 2 (350 mg, 1.15

mmol), and K₂CO₃(3.9 g, 28.75 mmol) gave ethyl 2-[6-(2-

naphthoylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-car-

boxylate (343 mg, 74% over two steps). m/z 403 [M þ H]^þ. Ethyl

2-[6-(2-naphthoylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-

5-carboxylate (343 mg, 0.85 mmol) was hydrolyzed with 6 M

NaOH (5 mL, 30 mmol) to give 2-[6-(2-naphthoylamino)-3-aza-

bicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylic acid 17a (167 mg,

52%) which was carried forward without purification. m/z

375 [M þ H]^þ. Following method D, 2-[6-(2-naphthoylamino)-3-

azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylic acid 17a (167 mg,

0.45 mmol), HOBt (83 mg, 0.54 mmol), EDC hydrochloride

(103 mg, 0.54 mmol), triethylamine (314 μL, 2.25 mmol), and

O-(1-isobutoxyethyl)hydroxylamine (309 μL, 2.25 mmol) gave

N-(1-isobutoxyethoxy)-2-[6-(2-naphthoylamino)-3-azabicyclo-

[3.1.0]hex-3-yl]pyrimidine-5-carboxamide (58 mg, 26%). m/z

490 [M þ H]^þ. N-(1-Isobutoxyethoxy)-2-[6-(2-naphthoylamino)-

3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxamide (58 mg, 0.12

mmol) was stirred in 4 M HCl in dioxane (60 mL, 0.29 mmol) at

room temperature for 5 min. The solvent was removed in vacuo and

the residue dried under reduced pressure to give N-hydroxy-2-{6-

[(naphthalene-2-carbonyl)amino]-3-aza-bicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxamide 18a (27 mg, 59%). m/z 390 [M þ H]^þ. ¹H

NMR (300 MHz, DMSO-d₆) δ: 11.05 (1H, br s), 9.01 (1H, br s), 8.74

(1H, d, J = 3.9 Hz), 8.69 (2H, s), 8.43 (1H, br s), 8.05-7.55 (4H, m),

7.55-7.66 (2H, m), 3.95 (2H, d, J = 11.6 Hz), 3.70-3.60 (2H, m),

2.70-2.64 (1H, m), 2.04 (2H, br s).

N-Hydroxy-2-{6-[(2-naphthylmethyl)amino]-3-azabicyclo[3.1.0]-

hex-3-yl}pyrimidine-5-carboxamide 21a. Following method A, tert-

butyl 6-aminoazabicyclo[3.1.0]hexane-3-carboxylate 8 (200 mg, 1.01

mmol), 2-naphthaldehyde (148 mg, 0.96 mmol), and sodium bor-

ohydride (61 mg, 1.62 mmol) gave tert-butyl 6-(naphth-2-ylmethyl)-

3-azabicyclo[3.1.0]hexane-3-carboxylate as a colorless oil which was

used in the subsequent step without purification. m/z 339 [M þ H]^þ.

Following method B, tert-butyl 6-(naphth-2-ylmethyl)-3-azabicyclo-

[3.1.0]hexane-3-carboxylate (0.96 mmol) was stirred in 4 M HCl

(2 mL) in dioxane at room temperature for 0.5 h to give N-naphth-2-

ylmethyl-3-azabicyclo[3.1.0]hexan-6-amine hydrochloride 19a which

was used in the next step without purification, m/z 239 [M þ H]^þ.

Following method C, N-naphth-2-ylmethyl-3-azabicyclo[3.1.0]-

hexan-6-amine hydrochloride (0.96 mmol), ethyl 2-(methylsul-

fonyl)pyrimidine-5-carboxylate 2 (221 mg, 0.96 mmol), and

potassium carbonate (1.32 g, 9.6 mmol) gave ethyl 2-[6-(naphth-2-

ylmethyl)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylate

2-(6-{[(6-Fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-

3-yl)-N-hydroxypyrimidine-5-carboxamide Trifluoroacetate 21r.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8675

Following method A, 6-fluoroquinoline-2-carbaldehyde 10 (265 mg,

1.51 mmol) was stirred in MeOH (10 mL) with tert-butyl 6-amino-3-

azabicyclo[3,1,0]hexane-3-carboxylate 8 (300 mg, 1.51 mmol) at

room temperature for 3 h. NaBH₄(91 mg, 2.42 mmol) was then

added in one portion and stirring continued for 10 min. The

reaction was then carefully quenched with 1 M NaOH (5 mL),

and stirring continued for 20 min. The mixture was then diluted

with H₂O (50 mL) and extracted with Et₂O (2 ꢀ 100 mL). The

combined organic layers were dried over MgSO₄and solvent

was removed in vacuo to give tert-butyl 6-{[(6-fluoroquinolin-

2-yl)methyl]amino}-3-azabicyclo[3.1.0]hexane-3-carboxylate as a

colorless oil which was used in the next step without purification.

m/z 358 [M þ H] ^þ. ¹H NMR (300 MHz, DMSO-d₆) δ: 8.30 (1H,

d, J = 8.7 Hz), 8.02 (1H, m), 7.79-7.61 (3H, m), 4.72 (1H, d, J =

6.5 Hz), 3.98 (2H, s), 1.78 (1H, s), 1.50 (2H, s), 1.31 (9H, s).

Following method B, tert-butyl 6-{[(6-fluoroquinolin-2-yl)methyl]-

amino}-3-azabicyclo[3.1.0]hexane-3-carboxylate (1.51 mmol) was

stirred in 4 M HCl in dioxane (3 mL) at room temperature for

30 min. Solvent was then removed in vacuo and the residue dried to

give N-[(6-fluoroquinolin-2-yl)methyl]-3-azabicyclo[3.1.0]hexan-

6-amine hydrochloride 19r which was used in the next step without

purification. m/z 258 [M^þþH]. Following method C, N-[(6-fluor-

oquinolin-2-yl)methyl]-3-azabicyclo[3.1.0]hexan-6-amine hydro-

chloride 19r (1.51 mmol) was stirred in a 1:1 v/v DMF/CH₃CN

(20 mL) with K₂CO₃(2.1 g, 15.1 mmol) at room temperature under

a nitrogen atmosphere for 10 min. Ethyl 2-(methylsulfonyl)pyri-

midine-5-carboxylate 2 (350 mg, 1.51 mmol) was then added and

the mixture stirred for a further 10 min. The reaction was the

quenched with H₂O (50 mL) and extracted with EtOAc (2 ꢀ 100 mL).

The combined organic layers were dried over MgSO₄and solvent

was removed in vacuo to give the ethyl 2-(6-{[(6-fluoroquinolin-2-

yl)methyl]amino}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-carbox-

ylate as an orange oil which was used in the next step without

purification. m/z 408 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆) δ:

8.71(2H,s),8.32(1H, d, J= 9.8 Hz), 8.07-7.95 (2H, m), 7.81-7.71

(2H, m), 4.72 (1H, d, J = 7.6 Hz), 4.23 (2H, q, J = 8.7 Hz), 3.53

(2H, d, J = 12 Hz), 3.33 (2H, s), 1.88 (1H, br s), 1.71 (2H, br s), 1.24

(3H, t, J = 8.7 Hz). Ethyl 2-(6-{[(6-fluoroquinolin-2-yl)methyl]-

amino}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-carboxylate was

stirred in 1 M NaOH/THF (7 mL/7 mL) at room temperature for

16 h. The mixture was the acidified to pH 3 with 1 M HCl which

caused a precipitate to form. This was filtered off and dried to give

2-(6-{[(6-fluoroquinolin-2-yl)methyl]amino}-3-azabicyclo[3.1.0]-

hex-3-yl)pyrimidine-5-carboxylic acid 20r (193 mg, 34% over

three steps) as a light yellow solid and used without purification.

m/z 380 [M þ H]^þ. Following method D, 2-(6-{[(6-fluoroquino-

lin-2-yl)methyl]amino}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-

5-carboxylic acid 20r (193 mg, 0.51 mmol) was stirred with EDC

hydrochloride (117 mg, 0.61 mmol) and HOBt (117 mg, 0.61 mmol)

in DMF (10 mL) at room temperature under a nitrogen atmosphere.

O-(1-Isobutoxyethyl)hydroxylamine (352 μL, 2.55 mmol) was then

added followed by triethylamine (355 μL, 2.55 mmol). The mixture

was stirred for 16 h, then diluted with H₂O (50 mL) and extracted

with CH₂Cl₂(2 ꢀ 100 mL). The combined organic layers were dried

over MgSO₄, and solvent was removed in vacuo. The residue was

purified by flash column chromatography [eluting with 0-10%

MeOH in CH₂Cl₂] to give 2-(6-{[(6-fluoroquinolin-2-yl)methyl]-

amino}-3-azabicyclo[3.1.0]hex-3-yl)-N-(1-isobutoxyethoxy)pyri-

midine-;5-carboxamide (130 mg, 52%) as a light yellow oil. m/z

2-(6-{[(6-fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-3-yl)-

N-hydroxypyrimidine-5-carboxamide trifluoroaceate 21r as a light

orange solid (25 mg, 19%). m/z395 [M þ H]^þ. ¹HNMR(300MHz,

DMSO-d₆) δ: 11.09 (1H, br s), 9.61 (1H, br s), 9.01 (1H, br s), 8.67

(2H, s), 8.48 (1H, d, J = 8.4 Hz), 7.88 (1H, dd, J = 9.3 Hz, 2.7 Hz),

7.75 (1H, td, J= 8.7 Hz, 3.0 Hz), 7.66 (1H, d, J= 8.4 Hz), 4.69 (2H,

bs), 3.88 (2H, d, J = 11.7 Hz), 3.60 (2H, d, J = 11.7 Hz), 2.30 (2H,

s,), 2.75 (1H, s).

N-Hydroxy-2-{6-[methyl(naphthalene-2-sulfonyl)amino]-3-aza-

bicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxamide 23: Step 1. Pre-

paration of N-(Tetrahydro-2H-pyran-2-yloxy)-2-{6-[(naphthalene-

2-sulfonyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxa-

mide 22. 2-{6-[(2-Naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hex-

3-yl}pyrimidine-5-carboxylic acid 14a (1.0 g, 2.4 mmol) was stirred

with EDC hydrochloride (556 mg, 2.9 mmol) and HOBt (392 mg,

2.9 mmol) in DMF at room temperature under a nitrogen atmo-

sphere. 2-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (1.4 g,

12 mmol) was then added followed by triethylamine (1.66 mL,

12 mmol), and the mixture was left to stir for 16 h. The mixture

was then diluted with H₂O and extracted with CH₂Cl₂. The

combined organic layers were dried (MgSO₄), and solvent was

removed in vacuo. The residue was purified by flash column

chromatography [eluting with 0-10% MeOH in CH₂Cl₂] to give

22 (1.11 g, 90%). m/z 511 [M þ H]^þ.

Step 2. Preparation of N-(Tetrahydro-2H-pyran-2-yloxy)-

2-{6-[methyl(naphthalene-2-sulfonyl)amino]-3-azabicyclo[3.1.0]-

hex-3-yl}pyrimidine-5-carboxamide. NaH (147 mg, 3.75 mmol)

was washed with heptanes and suspended in THF (10 mL).

N-(Tetrahydro-2H-pyran-2-yloxy)-2-[6-(naphthalene-2-sul-

fonylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxamide

22 (1.11 g, 2.2 mmol) was then added, followed by addition of

dimethyl sulfate (0.22 mL, 2.3 mmol). The mixture was stirred for

48 h and then poured into CH₂Cl₂(150 mL), and 2.4 M ammonium

chloride (50 mL) was added. The aqueous layer was further

extracted with CH₂Cl₂(150 mL). The combined organic layers were

dried (MgSO₄) and concentrated in vacuo to give an orange oil. This

was carried forward without purification.

Step 3. Preparation of N-Hydroxy-2-{6-[methyl(naphthalene-

2-sulfonyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carbox-

amide 23. To N-(tetrahydro-2H-pyran-2-yloxy)-2-{6-[methyl-

(naphthalene-2-sulfonyl)amino]-3-azabicyclo[3.1.0]hex-3-yl}pyri-

midine-5-carboxamide was added TFA/CH₂Cl₂/MeOH (5 mL,

1:1:1 v/v/v mixture). The solution was stirred at room temperature

for 2 h. The mixture was then concentrated under reduced pressure

and purified by reverse phase HPLC to yield 23 (34 mg, 3% yield

over two steps). m/z 440 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-

d₆) δ: 11.05 (1H, br s), 9.01 (1H, br s), 8.61 (2H, s), 8.52-8.49 (1H,

m), 8.28-8.22 (1H, m), 8.18 (1H, d, J = 8.7 Hz), 8.06 (1H, m),

7.82 (1H, dd, J=1.8, 8.6Hz), 7.66-7.84 (2H, m), 3.78 (2H, d, J=

11.8 Hz), 3.58 (2H, m), 2.77 (3H, s), 2.25 (2H, m), 1.55 (1H, m).

N-Hydroxy-2-[6-(quinolin-2-ylamino)-3-azabicyclo[3.1.0]hex-

3-yl]pyrimidine-5-carboxamide 25: Step 1. Preparation of tert-

Butyl 6-(quinolin-2-ylamino)-3-azabicyclo[3.1.0]hexane-3-carboxylate

24. tert-Butyl 6-aminoazabicyclo[3.1.0]hexane-3-carboxylate 8 (200

mg, 1.01 mmol) was combined with 2-chloroquinoline (328 mg, 2.02

mmol), and the two solids melted at 100 °Cfor16h. Themixturewas

then cooled and the residue purified by flash column chromatogra-

phy [eluting with, 0-3% MeOH/CH₂Cl₂] to give compound 24 as a

brown oil (320 mg, 97%). m/z 326 [M þ H]^þ.

1

495 [M þ H]. H NMR (300 MHz, DMSO-d₆) δ: 8.69 (2H, s),

Step 2. Preparation of (3-Azabicyclo[3.1.0]hex-6-yl)quinolin-

2-yl-amine. Following method B, tert-butyl 6-(quinolin-2-ylamino)-

3-azabicyclo[3.1.0]hexane-3-carboxylate (320 mg, 0.98 mmol) was

stirred in 4 M HCl in dioxane (2 mL) at room temperature under N₂

for 15 min. The solvent was then removed in vacuo and the

residue dried under high-vacuum to give crude (3-azabicyclo-

[3.1.0]hex-6-yl)quinolin-2-yl-amine (220 mg) which was used in

the next step without purification. m/z 226 [M þ H]^þ.

8.30 (1H, d, J = 9.8 Hz), 8.02 (1H, dd, J = 6.5, 9.8 Hz), 7.74 (1H,

dd, J = 3.3, 9.8 Hz), 7.67-7.60 (2H, m), 4.90 (1H, q, J = 5.4, 9.7

Hz), 4.01 (2H, br s), 3.63 (2H, s), 3.49 (2H, d, J = 10.1 Hz), 1.87

(1H, br s), 1.70 (2H, br s), 1.30 (3H, d, J = 5.4 Hz), 0.85 (3H, s),

0.83 (3H, s). 2-(6-{[(6-Fluoroquinolin-2-yl)methyl]amino}-3-azabi-

cyclo[3.1.0]hex-3-yl)-N-(1-isobutoxyethoxy)pyrimidine-5-carboxa-

mide (130 mg, 0.26 mmol) was stirred in CH₂Cl₂(2 mL) at room

temperature, and 4 M HCl in dioxane (130 μL, 0.52 mmol) added.

The mixture was stirred for 10 min and then the solvent removed in

vacuo. The residue was purified by preparative HPLC to give

Step 3. Preparation of Ethyl 2-[6-(Quinolin-2-ylamino)-3-azabi-

cyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylate. (3-Azabicyclo[3.1.0]

hex-6-yl)quinolin-2-ylamine (220 mg, 0.98 mmol) was stirred in

8676 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24

Moffat et al.

CH₃CN (10 mL) and DMF (10 mL) at room temperature under

N₂. Potassium carbonate (1.35 g, 9.8 mmol) was then added and

the mixture stirred for 15 min. Ethyl 2-(methylsulfonyl)pyrimidine-

5-carboxylate 2 (227 mg, 0.98 mmol) was then added to the

reaction mixture, and stirring continued for 30 min. The mixture

was then diluted with H₂O (100 mL) and extracted twice

with EtOAc (2 ꢀ 100 mL). The combined organic extracts were

dried (MgSO₄) and solvent was removed in vacuo to give ethyl

2-[6-(quinolin-2-ylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-

5-carboxylate as a light brown solid which was used in the next step

without purification. m/z 376 [M þ H]^þ.

bonyl)amino]methyl}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-car-

boxylate (1.7 g, 4.7 mmol) was stirred in CH₂Cl₂(20 mL) at room

temperature under a nitrogen atmosphere. Then 4 M HCl in

dioxane (2.35 mL, 9.4 mmol) was added, immediately causing a

solid to precipitate. The mixture was left to stir for 30 min, and then

the solvent was removed in vacuo. The residue was dissolved in

CH₂Cl₂(100 mL) and washed with saturated NaHCO₃(100 mL).

The organic layer was then dried (Na₂SO₄) and the solvent

removed in vacuo to give the title compound as an orange solid

(1.2 g, 97%). m/z 263 [M þ H]^þ. ¹H NMR (300 MHz, DMSO-d₆)

δ: 8.76 (2H, s), 4.26 (2H, q, J = 7.2 Hz), 3.82 (2H, d, J = 11.7 Hz),

3.55 (2H, d, J = 11.7 Hz), 3.33 (4H, m), 1.58 (2H, m), 1.29 (3H, t,

J = 7.2 Hz), 0.64 (1H, m).

Step 4. Preparation of 2-[6-(Quinolin-2-ylamino)-3-azabicyclo-

[3.1.0]hex-3-yl]pyrimidine-5-carboxylic Acid. Ethyl 2-[6-(quinolin-

2-ylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylate

(0.98 mmol) was stirred in THF (10 mL) and H₂O (10 mL) at

room temperature for 64 h. The mixture was then acidified to pH

3 using 1 M HCl and the solvent removed in vacuo to give a

brown solid. The solid was collected and washed with a little H₂O

to give the title compound as a brown solid (103 mg, 30% over

three steps). m/z 348 [M þ H]^þ.

Step 3. Preparation of 2-[6-(Aminomethyl)-3-azabicyclo[3.1.0]-

hex-3-yl]pyrimidine-5-carboxylic Acid 26. Ethyl 2-[6-(amino-

methyl)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxylate

(200 mg, 0.76 mmol) was stirred with 2-naphthaldehyde (119 mg,

0.76 mmol) in MeOH (10 mL) at room temperature under a

nitrogen atmosphere for 16 h. Sodium borohydride (46 mg, 1.22

mmol) was then added and the mixture stirred for 10 min.

Saturated aqueous NH₄Cl (20 mL) was added and the mixture

stirred for 20 min. The mixture was then diluted with H₂O (50 mL)

and extracted with Et₂O (2 ꢀ 100 mL). The combined organic

layers were dried (MgSO₄) and the solvent was removed in vacuo

to give the title compound as a yellow oil which was used in the next

step without purification. m/z 403 [M þ H]^þ.

Step 5. Preparation of N-(1-Isobutoxyethoxy)-2-[6-(quinolin-

2-ylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxyamide.

2-[6-(Quinolin-2-ylamino)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-

5-carboxylic acid (103 mg, 0.29 mmol) was stirred in DMF (10 mL)

at room temperature under a nitrogen atmosphere. EDC hydro-

chloride (67 mg, 0.35 mmol) and HOBt (47 mg, 0.35 mmol) were

added, and the mixture was stirred for 10 min. O-(1-Isobutox-

yethyl)hydroxylamine (200 μL, 1.45 mmol) and triethylamine

(202 μL, 1.45 mmol) were then added, and the mixture was stirred

for 16 h. The mixture was then diluted with H₂O (100 mL) and

extracted with CH₂Cl₂(2 ꢀ 100 mL). The combined organic

extracts were dried (MgSO₄), and solvent was removed in vacuo.

The residue was purified by column chromatography [eluting with

0-10% MeOH/CH₂Cl₂] to give the title compound as a white solid

(48 mg, 36%). m/z 463 [M þ H]^þ.

Step 4. Preparation of 2-(6-{[(Naphthalen-2-ylmethyl)amino]-

methyl}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-carboxylic Acid.

2-[6-(Aminomethyl)-3-azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-car-

boxylic acid 26 (0.76 mmol) was stirred in THF (6 mL) and 1 M

NaOH (6 mL) at room temperature for 16 h. The mixture was then

acidified to pH 3 with 2 M HCl, causing a solid to precipitate. This

was collected and dried to give the title compound as a white solid

(72 mg, 25% over two steps) which was used in the next step

without purification. m/z 375 [M þ H]^þ.

Step 6 Preparation of N-Hydroxy-2-[6-(quinolin-2-ylamino)-3-

azabicyclo[3.1.0]hex-3-yl]pyrimidine-5-carboxamide 25. N-(1-Iso-

butoxyethoxy)-2-[6-(quinolin-2-ylamino)-3-azabicyclo[3.1.0]hex-

3-yl]pyrimidine-5-carboxyamide (48 mg, 0.1 mmol) was stirred in

CH₂Cl₂(2 mL) at room temperature under a nitrogen atmo-

sphere. Then 4 M HCl in dioxane (20 μL, 0.2 mmol) was added,

immediatelycausing a solidtoprecipitate. Themixture was stirred

for 10 min and then solvent removed in vacuo. CH₂Cl₂(10 mL) was

added to the residue and the precipitate filtered and dried

to give compound 25 as a white solid (21 mg, 58%). m/z 363

Step 5. Preparation of N-(1-Isobutoxyethoxy)-2-(6-{[(naphthalen-

2-ylmethyl)amino]methyl}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-

5-carboxamide. 2-(6-{[(Naphthalen-2-ylmethyl)amino]methyl}-

3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-carboxylic acid (72 mg,

0.19 mmol) was stirred with EDC hydrochloride (44 mg, 0.23

mmol) and HOBt (31 mg, 0.23 mmol) in DMF (10 mL) at room

temperature under a nitrogen atmosphere for 10 min. O-(1-

Isobutoxyethyl)hydroxylamine (131 μL, 0.95 mmol) was then

added followed by triethylamine (132 μL, 0.95 mmol) and the

mixture allowed to stir for 64 h. The mixture was then diluted with

H₂O (50 mL) and extracted with CH₂Cl₂(2 ꢀ 100 mL). The

combined organic layers were dried (MgSO₄), and the solvent was

removed in vacuo. The residue was purified by column chroma-

tography [eluting with 0-10% MeOH/CH₂Cl₂] to give the title

compound as a colorless oil (43 mg, 46%). m/z 490 [M þ H]^þ.

Step 6. Preparation of N-Hydroxy-2-{6-[(naphthalen-2-ylmethyl-

amino)methyl]-3-azabicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxamide

27. N-(1-Isobutoxyethoxy)-2-(6-{[(naphthalene-2-ylmethyl) amino]-

methyl}-3-azabicyclo[3.1.0]hex-3-yl)pyrimidine-5-carboxamide

(43 mg, 0.09 mmol) was stirred in CH₂Cl₂(2 mL) at room

temperature under a nitrogen atmosphere, and 4 M HCl in dioxane

(45 μL, 0.18 mmol) was added. This immediately caused a solid to

precipitate. The mixture was allowed to stir for 10 min and then the

solvent was removed in vacuo to give the title compound 27 as a

white solid (16 mg, 50%). m/z 390 [M þ H]^þ. ¹H NMR (300 MHz,

DMSO-d₆) δ: 11.07 (1H, br s), 9.11 (2H, br s), 8.66 (2H, s), 8.03

(2H, m), 7.95 (2H, m), 7.68 (1H, m), 7.58 (2H, m), 4.34 (2H, m),

3.88 (2H, d, J=11.7 Hz), 3.55(2H, m), 2.99(2H, m), 2.50 (1H, m),

1.91 (2H, m).

1

[M þ H]^þ. H NMR (300 MHz, DMSO-d₆) δ: 12.96 (1H, br s),

11.12(1H,brs),10.24(1H,brs),8.33(1H,d,J=8.8Hz),8.28(2H,s),

8.09 (1H, d, J=8.0Hz),7.94(1H,d,J= 8.0 Hz), 7.81 (1H, t, J=7.6

Hz), 7.53 (1H, t, J = 7.6 Hz), 7.15 (1H, d, J = 8.8 Hz), 4.27 (2H, d,

J=11.7Hz), 3.63(2H, d,J=11.7Hz), 2.99(1H, m), 2.19(2H, brs).

N-Hydroxy-2-{6-[(naphthalen-2-ylmethylamino)methyl]-3-azabi-

cyclo[3.1.0]hex-3-ylpyrimidine-5-carboxamide 27: Step 1. Preparation

of Ethyl 2-(6-{[(tert-Butoxycarbonyl)amino]methyl}-3-azabicyclo-

[3.1.0]hex-3-yl)pyrimidine-5-carboxylate. Compound 12 (2.27 g,

10.7 mmol) was stirred in CH₃CN/DMF (60 mL, 1:1) with potas-

sium carbonate (4.44 g, 32.1 mmol) at room temperature under a

nitrogen atmosphere for 10 min. Ethyl 2-(methylsulfonyl)pyri-

midine-5-carboxylate 2 (2.48 g, 10.7 mmol) was then added and

the mixture stirred for 30 min. The mixture was then diluted with

H₂O (100 mL) and extracted with EtOAc (2 ꢀ 100 mL). The

combined organic extracts were dried (MgSO₄), and the solvent was

removed in vacuo. The residue was purified by column chromatog-

raphy [eluting with 0-5% MeOH in CH₂Cl₂] to give the title

1

compound as a white solid (3.5 g, 90%). m/z 363 [M þ H]^þ. H

NMR (300 MHz, DMSO-d₆) δ:8.76(2H,s), 6.92(1H,m),4.26(2H,

q, J = 7.2 Hz), 3.82 (2H, d, J= 11.7 Hz), 3.54 (2H, d, J = 11.7 Hz),

2.93 (2H, m), 1.61 (2H, m), 1.38 (9H, s), 1.28 (3H, t, J = 7.2 Hz),

0.70 (1H, m).

Acknowledgment. We thank Dr. Alan Drummond,

Dr. Alan Davidson (both of Chroma Therapeutics), and Pro-

fessor Paul Workman (Institute of Cancer Research) for their

help during the course of this work and in the preparation of this

Step 2. Preparation of Ethyl 2-[6-(Aminomethyl)-3-azabicyclo-

[3.1.0]hex-3-yl]pyrimidine-5-carboxylate. Ethyl 2-(6-{[(tert-butoxycar-

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8677

manuscript. We are grateful to Evotec (UK) Ltd. (assistance in

the preparation of intermediates and 15a), EpiStem Ltd. (in vivo

efficacy studies on 21r), and Dr. Michael Charlton (Chroma

Therapeutics, computational chemistry). The work of the Cancer

Therapeutics Unit was supported by Cancer Research UK Grant

Numbers C309/A2187, C309/A2874, C309/A8365, and C302/

A8265 and Infrastructure Support Grant Number C302/A7803

and by The Institute of Cancer Research. We acknowledge NHS

funding to the NIHR Biomedical Research Centre. The authors

from the Institute of Cancer Research (ICR) declare a conflict of

interest. The ICR has a commercial interest in the development of

HDAC inhibitors and operates a reward to inventors’ scheme.

The ICR has had a research collaboration with Chroma Ther-

apeutics and is a shareholder in the company, as is Cancer

Research Technology. Wynne Aherne has been a consultant to

Chroma Therapeutics.

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Supporting Information Available: Methods for fixed cell and

electrochemiluminescent ELISA; results showing effects of 21f and

21r on proliferation of a P-glycoprotein isogenic cancer cell line

pair; in vitro effects on histone and R-tubulin acetylation; in vivo

PK of 21f and effects on histone H3 and R-tubulin acetylation in

samples from a HCT116 human tumor mouse xenograft efficacy

study; synthesis and analytical data for 15b, 18b-c, 21b-e, and

21g-21q; single crystal X-ray structure of 21r. This material is

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

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(25) Equilibrium solubility was measured by resuspension of amor-

phous compound at 200 μg/mL in either 100% DMSO or 100%

phosphate buffer at pH 5.5 and 7.4. Phosphate buffered samples

were mixed for 24 h at room temperature using a PTFE magnetic

stirrer bar. DMSO samples were mixed for 24 h at room tempera-

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Article Doi

Discovery of 2-(6-{[(6-Fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex- 3-yl)- N -hydroxypyrimidine-5-carboxamide (CHR-3996), a class i selective orally active histone deacetylase inhibitor

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