Synthesis of N-heterocyclic ligands for use in affinity and mixed mode chromatography

Tetrahedron 67 (20 1 1 ) 471e485

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Tetrahedron

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Synthesis of N-heterocyclic ligands for use in afﬁnity and mixed mode

chromatography

,

*

Simon J. Mountford ^a, Eva M. Campi ^a, Andrea J. Robinson ^{a b}, Milton T.W. Hearn ^a

^aCentre for Green Chemistry, Monash University, Clayton 3800, Australia

^bSchool of Chemistry, Monash University, Clayton 3800, Australia

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 28 June 2010

Received in revised form 12 October 2010

Accepted 1 November 2010

Available online 12 November 2010

A set of heterocyclic ligands have been synthesised for use in the preparation of mixed mode afﬁnity

chromatographic adsorbents for application in the puriﬁcation of proteins, including antibodies. The

ligand structures were designed to consist of a pyridinyl or related aza-heterocyclic nucleus bearing

a pendant arm containing either an alkylamine, alkylthiol or hydroxyalkyl nucleophilic group to allow

their facile immobilisation onto an activated support matrix. Ligand diversity was achieved by altering

the length of the alkyl chain between the heterocyclic nucleus and nucleophilic group, varying the po-

sition of alkyl chain attachment to the heterocycle, and incorporating extra substituents into the pyr-

idinyl or related aza-heterocyclic ring. This diversity in ligand structure was intended to enable key

structural features of the ligand, required for efﬁcient protein binding, to be determined. In contrast to

the previously used multi-step procedures for the preparation of analogous substituted pyridine or aza-

heterocyclic compounds, the synthesis routes for the ligands described here have generally utilized very

mild, one-step reactions with readily available heterocyclic precursors.

Keywords:

N-Heterocyclic sulphanyl compounds

Nucleophilic substitution reactions

Chromatographic ligands

Chemical immobilisation

Afﬁnity chromatography

1. Introduction

chemical ligands, which can be immobilised onto suitable support

matrices and employed as a substitute for protein A in the puriﬁ-

Afﬁnity and mixed mode chromatographic methods are widely

utilized in the biological and biomedical sciences, e.g., for the pu-

riﬁcation of enzymes, proteins, oligo-nucleotides, DNA, including

single and double coiled plasmid DNA, RNA and cells.^1e3The

technique involves the separation of the target biomolecule from

other components in a complex mixture based on its ability to

undergo reversible interaction with a complementary bio-speciﬁc

ligand immobilised onto a support matrix. High speciﬁcity is ach-

ieved when the binding interaction replicates the same processes

that occur with naturally-occurring biorecognition events. The

various applications of afﬁnity and mixed mode chromatography

have been extensively discussed and reviewed in the literature.^4e8

Commercially important classes of proteins include antibodies,

which currently ﬁnd wide application in therapy and diagnosis.^9,10

One of the most commonly used methods for the puriﬁcation of

antibodies involves afﬁnity chromatography using immobilised

protein A (a surface protein originally found over 50 years ago in the

cell wall of the bacteria Staphylococcus aureus). However the use of

protein A based afﬁnity adsorbents has a number of well recognised

limitations.^11e13Accordingly, the search for low-molecular weight

cation of antibodies, has attracted attention over the past 20 years.

For example, Porath et al. have described a separation process

for the chromatographic fractionation of protein mixtures, which

they termed ‘thiophilic adsorption’.¹⁴These researchers docu-

mented the adsorption properties of several serum proteins, in-

cluding immunoglobulins (e.g., IgGs) and

several hydrophilic resins prepared by activation with divinylsul-

fone (DVS), followed by reaction with -mercaptoethanol. This

a2-macroglobulins, with

b

mode of adsorption is particularly affected by the presence of high

concentrations of neutral kosmotropic (water structuring) salts.

Subsequently, Porath and Oscarsson described a similar type of

binding behaviour with some proteins when 2-mercaptopyridine

was coupled to an epichlorohydrin (epoxy-) activated gel (Fig. 1).¹⁵

Although the immobilised 2-mercaptopyridine ligand was found to

O

S

N

OH

* Corresponding author. E-mail address: milton.hearn@monash.edu (M.T.W.

Hearn).

Fig. 1. 2-Mercaptopyridine coupled to an epichlorohydrin-activated gel.

doi:10.1016/j.tet.2010.11.003

472

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

adsorb

a

greater amount of IgG when compared to the

where the sulfur atom was replaced by an oxygen or nitrogen atom.

These alternatives, however, showed minimal adsorption of IgG,

again indicating the importance of the presence of the sulfur atom.

With the ever increasing need for more efﬁcient and cost ef-

fective protocols for the puriﬁcation of therapeutic monoclonal

antibodies, considerable motivation exists²⁴to develop other more

effective low-molecular weight molecules that are able to: (i) bind

antibodies with similar afﬁnity to protein A and (ii) possess im-

proved physical and chemical properties. Moreover, these synthetic

ligands should preferentially have characteristics that result in in-

creased resistance to degradation from chemical and biological

agents, lower toxicity, reduced leakage, high antibody binding ca-

pacities and broader selectivity. Overall, improvements in the de-

sign and synthesis of new molecules with these properties will lead

to signiﬁcantly reduced production costs for antibodies, particu-

larly therapeutic monoclonal antibodies.

Herein, we report the synthesis of a range of low-molecular

weight ligands based on several N-heterocycles containing a pri-

mary alkylamine group. Our synthetic objective was to prepare

these compounds in high purity by efﬁcient methods and to sub-

sequently immobilise them onto suitable solid phase support ma-

terials. Our preliminary results with these new afﬁnity adsorbents

indicate their potential for the static (batch) capture and chro-

matographic puriﬁcation of antibodies.

4-substituted derivative at comparable immobilisation densities,

the latter was more selective.¹⁶Moreover, when the 2-mercapto-

pyridine ligand was replaced by 2-aminopyridine or 2-hydroxy-

pyridine signiﬁcant reduction in adsorption capacity was observed,

clearly indicating the need for a sulfur atom to be present.

Similarly, Knudsen et al. have prepared a variety of thiophilic ad-

sorbents based on the immobilisation of heteroaromatic ligands onto

divinylsulfone-activated gels.¹⁷For example, isolation of IgG was

achieved from human serum in the presence of lyotropic salts using

afﬁnity adsorbents derived from 2-, 3- or 4-hydroxypyridine, 2-ami-

nopyridine, 4-aminobenzoic acid, 4-methoxyphenol and imidazole.¹⁷

Extending the work of Porath and Oscarsson¹⁴, Schwarz et al.

immobilised 2-mercaptopyridine, 2-mercaptopyrimidine and mer-

capto-thiazoline onto both epoxy-activated agarose and silica.¹⁸

These latter two ligands were chosen on the basis of their higher

hydrophilicity and a higher electron density compared to 2-mercap-

topyridine. Schwarz later prepared several adsorbents using ﬁve-

membered heterocyclic rings containing at least two heteroatoms¹⁹

whilst Scholz et al. coupled 2-mercaptopyridine, 2-mercaptopyr-

imidine and 2-mercaptonicotinic acid to DVS- and epichlorohydrin-

activated Sepharose.²⁰

When compared to protein A afﬁnity chromatography, these

thiophilic chromatographic materials potentially have a number of

advantages, including: (i) lower cost to produce the adsorbent; (ii)

broader speciﬁcity for binding to antibodies and other proteins

present in various feedstocks; (iii) the possibility to realize higher

binding capacities if high density immobilisation could be ach-

ieved; (iv) milder elution conditions; (v) greater chemical stability

of the immobilised ligands, (vi) possibility for reduced ligand

leakage under alkaline clean-in-place (CIP) conditions and (vii)

ability to separate and purify antibody fragments that lack a protein

A biorecognition site.

2. Results and discussion

The compounds prepared in this work retain a heterocyclic core

and exocyclic sulfur atom but in contrast to the ligands developed

earlier by Oscarsson and Porath¹⁶or other investigators, the novel

ligands described here utilize an aliphatic primary amine tether,

which enables both the very efﬁcient immobilisation of the ligand

onto a solid support material and simultaneously provides a spacer

group between the interactive core and the solid support. In this

manner, this pendant structure is attached to the heterocyclic ring

via the exocyclic sulfur atom and can be readily varied in structure

to allow the hydrophobicity of the ligand to be easily altered.

For example, the PSEA and PSPA series (6, Scheme 1) possess an

ethyl- or propyl-amine group extending from the exocyclic sulfur,

respectively. These derivatives were prepared by hydrazinolysis of

the corresponding phthalimides, based on modiﬁcations to the

procedures for the synthesis of N-alkylthiopyridine-benzo[b]thio-

phene-2-carboxamides or 4-(2-pyridylthio)butylamino-methyl-

enebisphosphonate and other amino-methylene-bisphosphonic

A further extension of the use of heteroaromatic ligands immo-

bilised onto support materials can be found in hydrophobic charge

induction chromatography (HCIC). This technique, ﬁrst described

by Burton and Harding,²¹takes advantage of the pH-dependent be-

haviour of ligands, such as 4-mercaptoethylpyridine (4-MEP). This

ligand has a greater speciﬁcity for immunoglobulins than 4-mer-

captoethylbenzene.²²In previous studies, this ligand has been

chemically attached to a hydrophilic cellulose-based resin which has

beenpreviouslyactivatedwithallylbromide. Theresultingadsorbent,

MEP HyperCel^Ò, is commercially available. Boschetti and Guerrier²³

have prepared a number of MEP-related ligands including ones

Scheme 1. General route for the preparation of the pyridinylsulfanylethyl- and propyl-amine compounds, 6.

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

473

acid derivatives described by Boschelli et al.²⁵and Sodha et al.,²⁶

respectively (route 1, Scheme 1).

and 2-aminoethanethiol hydrochloride 5 modelled on the method

originally described for the synthesis of 2-[[2-(dimethylamino)ethyl]

thio]-3-phenylquinoline by Blackburn et al.³⁰The 2-PSEA derivative

6a was obtained in 71% yield while the 4-PSEA derivative 6b was

acquired in an 87% yield (Table 2, entries i and ii). These yields are

superior to the <40% yield for the same derivatives prepared by the

earlier two step procedures³¹of route 1. Although the puriﬁcation of

4-pyridinylsulfanylethylamine, 6b, prepared via route 2, was

attempted by distillation under high vacuum, this resulted in de-

composition of the crude product and formation of two unidentiﬁed

compounds. However, the target 4-PSEA, 6b, could be readily puriﬁed

in very high isolated yield (87%) by column chromatography using

silica Si60 and dichloromethane/methanol/ammonium hydroxide

(9:1:0.1, v/v) as the eluent. Similar chromatographic methods were

also used with other substituted pyridinylsulfanylethylamines in

circumstances where attempted distillation lead to decomposition.

The 2- and 4-mercaptopyridines 1 were coupled to N-(2-bro-

moethyl)phthalimide 2, n¼1 or N-(2-bromopropyl)phthalimide 2,

n¼2 in DMF or reﬂuxing acetone in the presence of potassium

carbonate. The phthalimides

3 were puriﬁed by trituration,

recrystallisation or ﬂash chromatography and obtained in moderate

yields (Table 1). The phthalimides 3 were converted into the cor-

responding primary amines 6 by the use of hydrazine hydrate in

EtOH. The desired amines 6 were obtained in reasonable yields

(Table 1) after a basic (pH ∼9) work-up, with only the 4-PSPA 6d

requiring further puriﬁcation. All of the synthesized compounds

were characterised by ¹H and ¹³C NMR, FTIR, MS, and melting point

and by high resolution electrospray mass spectrometry and ele-

mental analysis for all new compounds. Since the two-step syn-

thesis described above led to only ca. 40% overall yield for both

Table 1

Preparation of pyridinyl sulfanyl ethylamines 6 (R¼H) via route 1 (Scheme 1)

Entry

6

1

2

Step 1

Step 2

Solvent

Temp

Time

2 h

Yield % 3

Time

Yield % 6

i

6a; 2-PSEA

n¼1

Acetone

Reﬂux

64

16 h

58

ii

6b; 4-PSEA

6c: 2-PSPA

6d; 4-PSPA

n¼1

n¼2

Reﬂux

rt

2 h

43

94

48

16 h

17 h

16 h

83

96

68

iii

iv

Acetone

4 h

DMF

3.5 h

Speciﬁc methods for the synthesis of these compounds can be found in the Experimental section. Literature data for known compounds can be found at the following citations:

3a;^26,273b;^26,283c;²⁶3d;²⁵6a;²⁹6b;²⁶6c;²⁶6d.²⁶

PSEA derivatives (Table 1, entries i and ii), alternative methods were

sought.

Preparation of 3-PSEA 6e via route 2 led to a 58% conversion of the

starting 3-bromopyridine to the product and an isolated yield of 50%

after puriﬁcation by ﬂash chromatography (Table 2, entry iii). Alter-

natively, the preparation of 3-PSEA 6e was achieved by ﬁrstly con-

verting the starting 3-bromopyridine to the N-oxide derivative in

order to facilitate nucleophilic attack at the 3-position of the ring. This

was achieved by the oxidation of 3-bromopyridine, based on the

oxidation of 2-bromopyridine with hydrogen peroxide in glacial

acetic acidasdescribed byEvans and Brown³²and Ochiai,³³giving the

N-oxide in a 70% yield without the need for puriﬁcation. 3-PSEA was

then prepared in a 38% yield by reacting 3-bromopyridine N-oxide

with 2-aminoethanethiol hydrochloride 5 followed by addition of

phosphorus trichloride based on a procedure for the preparation of

3-arylthio-pyridones described by Batmanghelich and Turner.³⁴

For synthesis of these compounds via route 2 (Scheme 1) it was

observed the yield was enhanced when a slurry of NaH in neat HMPA

was used, i.e., without the addition of THF. The preparation of the Me-

2-PSEA derivatives was achieved (via route 2, Scheme 1) in acceptable

yields of 38% and 63% for the 4-Me and 6-Me analogues 6f and 6g,

respectively, after work-up and puriﬁcation (Table 2, entries vi and v).

Preparation of Me-2-PSEA derivatives was initially attempted fol-

lowing the method described by Constable et al.;²⁹however, under

The preparation of 2-PSEA has previously been reported in a 77%

yield by Constable et al.,²⁹whereby 2-bromopyridine was allowed

to react with 2-aminoethanethiol hydrochloride 5 in a solution of

sodium ethoxide in EtOH at reﬂux for 24 h. The method in-

vestigated here (route 2, Scheme 1) also involves the reaction of

a halogenated pyridine and 5, however in this case the reaction

proceeded more rapidly under much milder conditions using NaH

in dry THF and/or hexamethylphosphoramide (HMPA) or DMF at rt

(Table 2). These conditions removed the possibility of the ethoxide

base acting as a nucleophile as observed earlier by Constable et al.²⁹

or by us during the preparation of 4-QSEA (see later discussion).

Furthermore, compared to route 1, route 2 also has the beneﬁt that

a larger range of substituted halogenated pyridines are more

readily available compared to the corresponding substituted mer-

capto-pyridines, allowing for more versatile preparation of a di-

verse library of derivatives with varying properties, e.g., ligands

with different hydrophobicity, steric bulk and electron-withdraw-

ing or electron-donating effects.

The ethylamines 6 were therefore synthesised in a one-step, one

pot reaction from commercially available halogenated pyridines 4

474

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

Table 2

Preparation of pyridinyl sulfanyl ethylamines 6 (R¼H, Me, Ph, OMe) via route 2 (Scheme 1)

Entry

6

4

Solvent

Temp

rt

Time

22 h

Yield % 6

i

6a; 2-PSEA

THF/HMPA

71

ii

6b; 4-PSEA

THF/HMPA

rt

2 days

87

iii

iv

6e; 3-PSEA

HMPA

rt

2 days

50

38

6f; 4-Mee2-PSEA

THF/HMPA

v

6g; 6-Mee2-PSEA

HMPA

rt

22 h

63

vi

6h; 2-Phe4-PSEA

6i; 6-Phe2-PSEA

HMPA

rt

3 h

82

vii

27 h

viii

ix

6j; 6-OMee2-PSEA

HMPA

DMF

rt

2 days

23 h

30

42

60 ^ꢀC

Speciﬁc methods for the synthesis of these compounds can be found in the Experimental section. Literature data for known compounds can be found at the following citations:

6a;²⁹6b,²⁶6e.³¹

these conditions the ¹H NMR spectra of the reaction mixture showed

only ca. 10% conversion to the desired product in each case.

To prepare the desired phenyl derivatives, commercially available

derivative 6g under the same conditions. The amine hydrochloride

salt of 6-OMe-2-PSEA 6j was prepared in a 39% yield by the addition

of a saturated solution of HCl in ether. Basiﬁcation afforded the free

amine in a 30% overall yield (Table 2, entry viii). This yield was

increased to 42% when the reaction was performed in DMF at 60 ^ꢀC

(Table 2, entry ix).

Electron withdrawing substituents positioned meta with respect

to the nucleophilic substitution position do not participate in reso-

nance stabilisation of the intermediate and therefore do not assist

substitution.Preparationof4-NO₂-2-PSEA 6kwas, however, achieved

via the N-oxide 7 in a one-pot synthesis (Scheme 2). The starting

chloro-pyridine N-oxide 7 was reacted with the carbamate 8 in

a suspension of NaH inTHF before deprotection of the BOC group and

reduction of the N-oxide with a solution of PCl₃in CHCl₃. The target

4-NO₂-2-PSEA 6k was obtained in ca. 68% yield and 95% purity. A

sample of the product was puriﬁed by preparative TLC (silica Si60) for

characterisation. In order to minimise the possible displacement of

the 4-nitro group as well as the 2-chloro group, a slight excess of the

pyridine N-oxide 7 was used in respect to the carbamate 8.

2-phenylpyridine was chlorinated at the a- and g-positions of the

pyridine ring, again via the N-oxide, and obtained in a 72% yield by a

procedure modelled on the synthesisby Zhanget al.³⁵of the 4-pyridyl

substituted tri-dentate bis[2-(2-pyridyl)ethyl]methylamine ligands.

Chlorination and subsequent reduction of the N-oxide was achieved

via a method similar to that described for the preparation of furo[2,3-

b/c]pyridine N-oxides by Shiotani and Taniguchi.³⁶Puriﬁcation by

column chromatography of the reaction mixture afforded the 2-

chloro-6-phenylpyridine (52%), 4-chloro-2-phenylpyridine (24%) and

5-chloro-2-phenylpyridine (0.8%). The 2- and 4-chloro products were

then reacted with 2-aminoethanethiol hydrochloride 5 (route 2,

Scheme 1), with both of the phenyl derivatives 6h and 6i obtained in

82% yield after chromatography (Table 2, entries vi and vii).

The electron-donating effect of the methoxyl group decreased

the reactivity of the meta-positioned chlorine substituent, resulting

in longer reaction times when compared to the 6-Me-2-PSEA

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

475

Scheme 2. Preparation of 4-NO₂e2-PSEA.

Preparation of 4-NO₂-2-PSEA 6k was originally attempted using

a procedure based on route 2 (Scheme 1). The product was isolated

as the HCl salt and the free amine regenerated to give a 36% yield of

2-Cl-4-PSEA rather than 4-NO₂-2-PSEA, i.e., substitution of the NO₂

group occurred in preference to the chloro group. This result is

consistent with other literature precedents, and indicated that

nucleophilic attack would be preferentially favoured at the carbon

preparation of various pyridine derivatives 6 (route 2, Scheme 1) can

be utilized to prepare related compounds from other heteroaryl

chlorides. Thus, a range of different heteroaryl sulfanyl ethylamines

10 were prepared from readily available heteroaryl chlorides as

shown in Scheme 3 with the results summarized in Table 3.

bearing the halogen (a-position) compared to the carbon bearing

the nitro group (

g

-position), if the electronic properties of the

pyridyl ring were ﬁrstly converted to the N-oxide derivative.³⁷

Based on the above synthetic methodologies, a range of pyr-

idinylsulfanylethylamines 6 have been prepared under mild condi-

Scheme 3. General route for preparation of sulfanyl ethylamines 10.

Table 3

Preparation of heteroaryl sulfanyl ethylamines 10 via Scheme 3

Entry

10

9

Solvent

HMPA

Temp

rt

Time

1 h

Yield % 10

i

10a; 4-QSEA

52

ii

10b; 2-QSEA

HMPA

rt

2 h

84

iii

iv

10c; 1-IQSEA

HMPA

rt

2.5 h

3 h

91

70

10d; 2-PymSEA

v

10e; 2-PyzSEA

10f; 4-TerPSEA

HMPA

DMF

rt

3 h

85

95

vi

50 ^ꢀC

3 h

tions from readily available halogenated pyridines 4. The generally

good yields obtained make this route (route 2, Scheme 1) preferable

when compared to the alternative two step route from pyridine

thiols 1 (route 1, Scheme 1). The one pot route to the 4-nitro com-

pound 6k (Scheme 2) illustrates the utility of N-oxides in this mode

of nucleophilic substitution chemistry with strongly electron-

withdrawing substituents. Moreover, the route employed above for

For example, the 4-quinoline derivative (4-QSEA, 10a) was

prepared as described in Scheme 3 and isolated as the amine salt

(71%) before conversion to the free base (52%) (Table 3, entry i).

Puriﬁcation of the free base was attempted by vacuum distillation,

however the product decomposed on heating. An alternative

method for the preparation of this compound modelled on the

procedure of Constable et al.²⁹for the synthesis of 2-PSEA with

476

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

ethanolic sodium ethoxide gave complete conversion of the start-

ing material into a 60:40 ratio of 4-QSEA and 4-ethoxyquinoline.

Separation by column chromatography led to the isolation of 4-

ethoxyquinoline (30%) but failed to yield 4-QSEA in a high purity

state. Subsequent trituration of the impure 4-QSEA fraction facili-

tated easy isolation of a new product N-[2-(quinolin-4-ylsulfanyl)-

ethyl]-quinolin-4-amine (11%). The 2-quinoline derivative (2-QSEA,

10b) and the 1-iso-quinoline derivative (1-IQSEA, 10c) were also

prepared as one pot reactions by the method described in Scheme

3. Both products were puriﬁed by precipitation of the amine salt

with subsequent conversion to the free base affording excellent

yields of 2-QSEA and 1-IQSEA (84% and 91%, respectively) (Table 3,

entries ii and iii). A small sample of 2-QSEA was also puriﬁed by

column chromatography and found to be stable, unlike 4-QSEA.

The preparation of 2-PymSEA 10d was achieved in 70% yield

after puriﬁcation by column chromatography while 2-PyzSEA 10e

was obtained in 85% yield (Table 3, entries iv and v). Similarly, 4-

TerPSEA³⁸10f was prepared via the method outlined in Scheme 3

using DMF as the solvent and obtained in a 95% yield with no

column chromatographic puriﬁcation required. The starting 4⁰-

chloro-2,2⁰:6⁰,2⁰⁰-terpyridine derivative was prepared as described

by Constable and Ward³⁹in an overall yield of 52% from ethyl

pyridine-2-carboxylate.

MPP 13b, which was obtained in a 10% yield after column chro-

matography (Table 4, entry ii). In addition, the disulﬁde dimer, 4,4⁰-

(disulfanediyldipropane-3,1-diyl)di-pyridine, formed by oxidation

of 4-MPP during the chromatographic step, was also isolated (4%).

Lithium diisopropylamide (LDA) was also used in the preparation

of the 3-derivative to prevent nucleophilic attack of the butyl group,

which had occurred during the preparation of 4-MPP. A low yield of

3% was obtained for 3-MPP (Table 4, entry iii) due to incomplete

conversion of the starting materials and formation of multiple side

products, which necessitated a two step puriﬁcation process in-

volving distillation and column chromatography. Although the one

pot route utilizing thiirane gave low yields of the 3- and 4-MPP, the

new 2-isomer 13a was obtained in 45% yield. This yield compares

favourably to the ca. 40% yield obtained for the two step preparation

of 3- and 4-MPP using H₂S as described by Furukawa et al.⁴⁰

The PMSEA derivatives 15 were prepared based on methods used

by Tilley et al.⁴²for the synthesis of N-(heterocyclic alkyl)pyrido[2,1-

b]quinazoline-8-carboxamides or Carson et al.⁴³for the synthesis

of (E)-3-(4-oxo-4H-quinazolin-3-yl)-2-propenamides, as shown in

Scheme 5. The desired picolyl chloride hydrochloride 14 was reacted

with 2-aminoethanethiol hydrochloride 5 in a solution of NaOH in

ethanol. The crude products were puriﬁed by column chromatogra-

phy to give verygood yields of all three isomers (Table 5, entries ieiii).

The preparation of propanethiol pyridines (MPP series) has

previously been described by Furukawa et al.⁴⁰and involved con-

version of commercially available hydroxyl derivatives into the

chloro derivatives and ﬁnally to the desired thiols. An alternative

one-step synthesis was investigated here and involved the reaction

of ethylene sulﬁde (thiirane) and the desired picoline 11 (Scheme 4).

This synthesis was achieved based on a method described for the

preparation of 2,2⁰-hydroxymethyl-substituted 4,4⁰-bipyridines by

Leighton and Sanders, who employed ethylene oxide to prepare the

corresponding propyl alcohol derivative.⁴¹

Scheme 5. General route for the preparation of PMSEA derivatives.

Table 5

Preparation of PMSEA 15 and PESEA 17 derivatives

Entry 15 or 17

14 or 16

Solvent Temp Time Yield %

S

Base

15 or 17

SH

THF

-

N

CH

N

CH

2

3

i

15a; 2-PMSEA

EtOH

rt

2.5 h 89

2.5 h 75

11

12

13

Scheme 4. General route for the preparation MPP derivatives.

The thiols were prepared by ﬁrstly generating the anion 12 of

the starting picoline 11 with either butyllithium or lithium diiso-

propylamide (LDA), a more hindered base, and then adding thiirane

at ꢁ78 ^ꢀC. The results are summarized in Table 4. The 2-MPP 13a

was obtained in a 45% yield after puriﬁcation by distillation (Table

4, entry i). The crude product obtained for the 4-derivative was

distilled to give a side product, 2-n-butyl-4-methylpyridine and 4-

ii

15b; 3-PMSEA

15c; 4-PMSEA

17a; 2-PESEA

iii

iv

EtOH

rt

2.5 h 94

Table 4

Preparation of the MPP derivatives 13

Reﬂux 2.5 h 95

Entry 13

11

Solvent Temp Time Base

Yield

% 13

i

13a; 2-MPP

THF

rt

o/n

n-BuLi 45

v

17b; 4-PESEA

17c; 3-PESEA

EtOH

Reﬂux 2.5 h 82

ii

13b; 4-MPP

13c; 3-MPP

n-BuLi 10

vi

Reﬂux 2.5 h

0

iii

THF

rt

o/n

LDA

3

Speciﬁc methods for the synthesis of these compounds can be found in the

Experimental section. Literature data for known compounds can be found at the

following citations: 15a;⁴⁷15b;⁴²15c;⁴³17a.⁴⁴

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

477

The PESEA derivatives 17 were synthesised based on a method

described by Kaasjager et al.⁴⁴for the preparation of 1-(2⁰-pyridyl)-

3-thia-5-aminopentane as shown in Scheme 6. The desired vinyl-

pyridine 16 was reacted with 2-aminoethanethiol hydrochloride 5

in a solution of NaOH in ethanol to give good yields of the 2- and 4-

derivatives (Table 5, entries iv and v). Only the 4-derivative 17b

required puriﬁcation (by column chromatography) to remove the

starting compound, 4-vinylpyridine. While the 2- and 4-vinyl-

pyridines were commercially available, 3-vinylpyridine was pre-

pared in 32% yield, based on a procedure described by Alunni

et al.⁴⁵for the preparation of isomeric 2-ﬂuoroethyl-pyridines, in-

volving a Wittig reaction of 3-pyridinecarboxyaldehyde with

methyltriphenylphosphonium bromide. The preparation of 3-

PESEA 17c using the method described above was not successful,

possibly due to the lack of resonance stabilisation of an in-

introduced ligand. As a consequence, adsorbents derived from

these epichlorohydrin-activated gels can be re-used for multiple

cycles without signiﬁcant loss of performance provided feedstock

fouling due to host cell components is not signiﬁcant. In the present

studies this favourable attribute was manifested, when the adsor-

bents were recycled, showing no signiﬁcant loss of the immobilised

ligand for at least 10 cycles following batch regeneration with 0.5 M

NaOH. As shown in Tables 6 and 7 several of these resins (selected

on the basis of their ligand density and the magnitude of their Q_m

values) could be obtained with ligand densities in the range of

282e346 mmol/g adsorbent as determined by nitrogen elemental

analysis. Immobilised ligand densities in this range were antici-

pated to result in high static Q_maxvalues (as mg mAb bound per

mL), indicative of high capacities for mAbs and suitability for dy-

namic chromatographic use. In comparison, the remaining immo-

bilised ligands either showed lower binding efﬁciencies or lower

overall recovery. With regard to the ﬁve exemplars shown in Tables

6 and 7, regeneration studies showed that these adsorbents were

relatively stable to clean-in-place (CIP) treatments with 0.5 M

NaOH during the regeneration stages whilst high mAb binding

capacities were retained when employed in the dynamic recycling/

re-use chromatographic mode of operation.

termediate formed following attack of the thiol at the

the 3-substituted pyridine ring.

b-carbon of

Table 6

Scheme 6. General route for the preparation of PESEA derivatives.

Static binding capacity of several adsorbents, following immobilisation of the ligand

onto epichlorohydrin-activated Sepharose 6 Fast Flow (Sephe). The Q_maxand R²

values were derived from the ﬁt of the experimental data to a ﬁrst order Langmuir

isotherm

The ﬁnal compound prepared to complete the ligand set was 2-

PSEOH 19, the alcohol terminated analogue of the 2-PSEA de-

rivative 6a, which contains a terminal primary amine. The alcohol

was prepared by reacting 2-mercaptoethanol 18 and 2-bromopyr-

idine in a slurry of NaH and HMPA at 40 ^ꢀC (Scheme 7). An alter-

native strategy to achieve the S_N1 nucleophilic displacement of the

halo-atom by a sulfur anion has been employed by Beugelmans

et al. for the preparation of benzothiophenes and thienopyr-

idines.⁴⁶Puriﬁcation of the crude product by column chromatog-

raphy gave the alcohol 19 in ca. 72% yield (and ca. 95% purity). A

similar reaction at ambient temperature gave only 54% conversion.

Adsorbent

Ligand Density

mol/g

Q_{max, static}

mAb

R

²(Langmuir

Isotherm)

m

Bound mg/

mL

Sephe2-QSEA

Sephe4-NO₂e2-PSEA

Sephe2-PSEA

Sephe2-PSPA

Sephe4-QSEA

329

307

282

346

55.6

52.8

24.7

28.0

55.3

0.990

0.981

0.988

0.966

0.990

Table 7

Dynamic binding capacity of several adsorbents for the mAb, following immobili-

sation of the corresponding ligand onto epichlorohydrin-activated Sepharose 6 Fast

Flow (Sephe), and the corresponding mAb recovery from the eluted peak

Adsorbent

Ligand Density

mol/g

Q_{max, dynamic}mAb

Bound mg/mL

Recovery (%)

m

Scheme 7. Preparation of 2-PSEOH.

Sephe2-QSEA

Sephe4-NO₂e2-PSEA

Sephe2-PSEA

Sephe2-PSPA

Sephe4-QSEA

329

307

282

346

26.9

52.8

24.7

28.0

55.3

87

91

90

91

90

Based on the above synthetic procedures, a variety of N-het-

erocyclic sulphanyl compounds with alkyl side chains bearing

a terminal primary amine, alcohol or thiol group were thus pre-

pared. Changes to the ligand structure and properties were ach-

ieved by including various ring substituents. In associated

experiments, these compounds were immobilised, using epichlo-

rohydrin-activated Sepharose Fast Flow^Òas the model solid phase

support material with the objective to evaluate their potential as

novel mixed mode afﬁnity chromatographic adsorbents for the

binding and puriﬁcation of monoclonal antibodies (mAbs) in both

static (batch) or dynamic (column) modes.

The choice of epichlorohydrin for the activation of the agarose-

based Sepharose Fast Flow^Òwas based on the well documented

observation^48,49that stable OeC bonds are generated during the

activation stage with polysaccharide-related polymers rich in hy-

droxyl groups, such as agarose or cellulose, whilst similarly stable

CeN bonds are formed following the nucleophilic opening of the

introduced epoxide functionality by the amino group of the

Moreover, as evident from Fig. 2, when used in the chromato-

graphic mode efﬁcient and selective capture, elution and column

regeneration could be achieved for the puriﬁcation of the target

antibody.

As anticipated, the maximum binding capacities determined by

static binding experiments were greater that the binding capacity

achieved under the working conditions of a dynamic chromato-

graphic separation. Nevertheless, these dynamic binding capacities

(25e52 mg/mL) compare very favourably with other mAb binding

chromatographic adsorbents, such as protein A Sepharose Fast

Flow^Ò(15e20 mg/mL)⁵⁰or MEP HyperCel^Ò(30e35 mg/mL).⁵¹As

such, these studies indicate that this new class of heterocyclic li-

gand may have signiﬁcant potential in the puriﬁcation of mAbs and

possibly other proteins. To this end, further investigations are un-

derway documenting this potential with feedstocks of greater

478

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

Fig. 2. Elution proﬁle for the chromatographic fractionation of a cell culture derived mAb utilizing the Sephe2-QSEA adsorbent. The column was equilibrated with Buffer A (25 mM

Tris, 600 mM Na₂SO₄, pH 9.0), loaded from the time interval (a) to (b) with a medium sample (50 mL) containing the cell culture derived mAb and then washed from the time

interval (b) to (c) solely with Buffer A (15 mL). The bound mAb was eluted by a step change at (c) with Buffer B (10 mL) (25 mM Hepes, pH 7.0). The column was then regenerated by

washing from (d) with 0.5 M NaOH (5 mL), and at (e) with Buffer B (5 mL) prior to re-use. A ﬂow rate of 1 mL/min was employed throughout. The monomeric mAb (as assessed by

SDS-PAGE analysis) was eluted in fraction no’s 16e18 with recovery of 87%, whilst the aggregated mAb (∼5% recovery) eluted in fraction no. 20. The solid line is the absorbance at

280 nm and the dashed line is the conductivity (mScm^ꢁ1).

complexity in composition and with scaled up chromatographic

systems. These additional results will be presented subsequently

once these ongoing investigations are completed.

3.2. General procedure (A) for preparation of phthalimide

derivatives: preparation of 2-[2⁰-(pyridin-2⁰⁰-sulfanyl)ethyl]-

1H-isoindole-1,3(2H)-dione (3a)

3. Experimental

3.1. General

The phthalimides 3aed were prepared by modifying the pro-

cedure outlined by Boschelli et al.²⁵as described below for the

preparation of phthalimide 3a.

N-(2-Bromoethyl)phthalimide (6.86 g, 27.0 mmol) and K₂CO₃

(6.84 g, 49.5 mmol) were added to a solution of 2-mercaptopyridine

(2.50 g, 22.5 mmol) in dry acetone (75 mL). The resulting mixture was

stirred at reﬂux for 2 h and then allowed to cool to ambient temper-

ature. The solid was collected by ﬁltration and washed with EtOAc.

The ﬁltrate was concentrated in vacuo to give a brown oil. A pre-

cipitate formed on standing and the resulting mixture was triturated

with 40% EtOAc/hexane to afford the title compound 3a as a white

solid (4.10 g, 64%), mp 94e96 ^ꢀC (lit.²⁶mp 98e99 ^ꢀC). n_max(KBr):

3046w, 1770m, 1713s, 1576s, 1455m, 1396s, 1359s, 1237w, 1127m,

Nuclear magnetic resonance spectra were recorded at 300 MHz

(¹H) or 75 MHz (¹³C) with a Bruker DPX-300 spectrometer or at

400 MHz (¹H) or 100 MHz (¹³C) with a Bruker DRX-400 spec-

trometer. The NMR spectra refer to solutions in deuterated base-

washed (Na₂CO₃) CDCl₃where tetramethylsilane (TMS) was used as

the internal standard (

solvent peak used as an internal reference in ¹³C NMR spectra.

Chemical shift values ( ) are given in parts per million (ppm) rel-

d

0.00 ppm) for ¹H spectra and the residual

d

ative to the residual solvent peak (or TMS) and coupling constants

are given in hertz (J Hz). Infrared spectra were recorded on a Per-

kineElmer 1600 series Fourier Transform infrared spectrometer.

Low resolution electrospray ionisation mass spectra (in positive

(ESI^þ) mode) were recorded on a Micromass Platform II API QMS

Electrospray mass spectrometer. High-resolution electrospray mass

spectra (HRMS) were recorded on a Bruker BioApex 47e Fourier

Transform mass spectrometer. Melting points were determined

using a Gallenkamp melting point apparatus with a digital ther-

mometer and are uncorrected. Tetrahydrofuran (THF) was distilled

from lithium aluminium hydride, stored over sodium wire and

distilled from sodium and benzophenone prior to use. Reagents and

compounds were purchased from SigmaeAldrich, Castle Hill, NSW,

Australia. Microanalyses were performed by The Campbell Micro-

analytical Laboratory, Department of Chemistry, University of

Otago, Dunedin, New Zealand.

1084m, 977w, 862w, 765s, 713s cm^ꢁ^{1 1}H NMR (200 MHz, CDCl₃):

.

d

3.52 (t, J¼6.6 Hz, 2H, H2⁰); 4.06 (t, J¼6.6 Hz, 2H, H1⁰); 6.93 (ddd,

J¼7.3, 4.9, 1.1 Hz, 1H, H5⁰⁰); 7.17 (apparent dt, J¼8.1, 1.0 Hz, 1H, H3⁰⁰);

7.44 (ddd, J¼8.0, 7.4, 1.9 Hz, 1H, H4⁰); 7.67e7.85 (m, 4H, H4, H5, H6,

H7); 8.36 (ddd, J¼4.9, 1.8, 0.9 Hz, 1H, H6⁰). ¹³C NMR (50 MHz, CDCl₃):

d

28.6 (C2⁰); 37.9 (C1⁰); 119.9 (C5⁰⁰); 122.7 (C3⁰⁰); 123.6 (C4, C7); 132.5

(C3a, C7a); 134.2 (C5, C6); 136.3 (C4⁰⁰); 149.8 (C6⁰⁰); 158.0 (C2⁰⁰); 168.5

(C1, C3). Mass spectrum (ESI^þ): m/z 284.9 (MþH)^þ(100%). The ¹H

NMR spectral data were consistent with literature data.²⁷

3.2.1. 2-[2⁰-(Pyridin-4⁰⁰-sulfanyl)ethyl]-1H-isoindole-1,3(2H)-dione

(3b). General procedure (A) was followed using N-(2-bromoethyl)

phthalimide (6.86 g, 27.0 mmol) and 4-mercaptopyridine (2.50 g,

22.5 mmol) in dry acetone (125 mL) to give a brown semi-solid

(8.45 g). The slurry was triturated with 30% EtOAc/hexane followed

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

479

by acetone/hexane to afford the title compound 3b as a light brown

solid (2.78 g, 43%), mp 143e145 ^ꢀC (lit.²⁶mp 147e148 ^ꢀC). n_max

(KBr): 3035w, 1767m, 1713s, 1576s, 1456m, 1397s, 1356m, 1188w,

1084m, 974m, 861w, 805m, 717s, 531w cm^ꢁ1. ¹H NMR (300 MHz,

was basiﬁed (pH 11) with 2 M NaOH solution and extracted with

EtOAc (3ꢂ40 mL). The combined organic extract was dried

(MgSO₄), ﬁltered and evaporated to give a pale yellow solid (0.56 g).

Analysis of the crude product by ¹H NMR spectroscopy revealed

a 4:1 mixture of the desired amine 6a and a side product. The crude

product was re-suspended in dry EtOH (25 mL) and additional

hydrazine hydrate was added (0.15 mL). The reaction was stirred at

ambient temperature and progress monitored by TLC. After 18 h,

additional hydrazine hydrate (0.30 mL) was added and the mixture

stirred for a further 8 h. The reaction was worked up as described

above to yield the title compound 6a as a yellow oil (0.48 g, 58%).

n_max(neat): 3363w, 3287w, 3044w, 2925m, 2862w, 1579s, 1556s,

1455s, 1415s, 1283m, 1124s, 1044w, 986m, 854bm, 759s,

CDCl₃):

d

3.29 (t, J¼7.3 Hz, 2H, H2⁰); 3.99 (t, J¼7.4 Hz, 2H, H1⁰); 7.25

(d, J¼5.9 Hz, 2H, H3⁰⁰, H5⁰⁰); 7.72e7.87 (m, 4H, H4, H5, H6, H7); 8.41

(d, J¼6.0 Hz, 2H, H2⁰⁰, H6⁰⁰). ¹³C NMR (75 MHz, CDCl₃):

d

28.8 (C2⁰);

37.0 (C1⁰); 121.2 (C3⁰⁰, C5⁰⁰); 123.7 (C4, C7); 132.1 (C3a, C7a); 134.5

(C5, C6); 147.6 (C4⁰⁰); 149.8 (C2⁰⁰, C6⁰⁰); 168.2 (C1, C3). Mass spec-

trum (ESI^þ): m/z 285.0 (MþH)^þ(100%). The ¹H NMR spectral data

were consistent with literature data.²⁸

3.2.2. 2-[3⁰⁰-(Pyridin-2⁰⁰-ylsulfanyl)propyl]-1H-isoindole-1,3(2H)-dione

(3c). General procedure (A) was followed using N-(3-bromopropyl)

phthalimide (6.63 g, 24.7 mmol), K₂CO₃(6.84 g, 49.5 mmol) and 2-

mercaptopyridine (3.03 g, 27.2 mmol) in dry acetone (75 mL) at

reﬂux for 4 h to give a light brown solid (10.5 g). The solid was trit-

urated with hexane and column chromatography of the residual solid

(SiO₂, hexane/EtOAc,1:1) gave the title compound 3c as a light yellow

solid (6.94 g, 94%), mp 100e101 ^ꢀC (lit.²⁶mp 103e104 ^ꢀC). n_max(KBr):

3451w, 2935w, 1763m, 1707s, 1612w, 1575s, 1456m, 1436m, 1393s,

1348m, 1316m, 1287m, 1244w, 1188w, 1123m, 1103m, 986m, 909w,

883m, 767m, 724s, 618w, 529 m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

724m cm^ꢁ1. ¹H NMR (400 MHz, CDCl₃):

d 1.60 (br s, 2H, NH₂); 3.00

(t, J¼6.2 Hz, 2H, H1); 3.29 (t, J¼6.4 Hz, 2H, H2); 6.97 (ddd, J¼7.4, 4.9,

1.1 Hz, 1H, H5⁰); 7.19 (apparent dt, J¼8.1, 1.0 Hz, 1H, H3⁰); 7.46 (ddd,

J¼8.1, 7.4, 1.9 Hz, 1H, H4⁰); 8.40 (ddd, J¼4.9, 1.9, 1.0 Hz, 1H, H6⁰). ¹³C

NMR (75 MHz, CDCl₃):

d

34.1 (C2); 41.9 (C1); 119.5 (C5⁰); _þ122.6

(C3⁰); 136.0 (C4⁰); 149.5 (C6⁰); 158.7 (C2⁰). Mass spectrum (ESI ): m/

z 154.7 (MþH)^þ(100%). The spectral data for 6a were consistent

with literature data.²⁹

2-(Pyridin-2⁰-ylsulfanyl)ethanamine (6a) was also prepared

following General procedure (A) as described in Section 3.4 using

2-bromopyridine (2.00 g, 12.7 mmol) to give a brown oil (1.54 g).

Column chromatography (SiO₂, CH₂Cl₂/MeOH/NH₄OH, 9:1:0.1)

gave the title compound 6a as a yellow oil (0.95 g, 49%). The

spectral data were consistent with those given above.

d

2.11 (m, 2H, H2⁰); 3.21 (t, J¼7.3 Hz, 2H, H3⁰); 3.84 (t, J¼6.9 Hz, 2H,

H1⁰); 6.92 (ddd, J¼7.3, 4.9, 1.1 Hz, 1H, H5⁰⁰); 7.14 (dt, J¼8.1, 1.0 Hz, 1H,

H3⁰⁰); 7.43 (ddd, J¼8.1, 7.3, 1.9 Hz, 1H, H4⁰⁰); 7.71 (m, 2H, H5, H6); 7.83

13

(m, 2H, H4, H7); 8.30 (ddd, J¼4.9, 1.9, 1.0 Hz, 1H, H6⁰⁰). C NMR

(75 MHz, CDCl₃): d

27.3 (C3⁰); 28.9 (C2⁰); 37.3 (C1⁰); 119.5 (C5⁰⁰); 122.5

(C3⁰⁰); 123.3 (C4, C7); 132.3 (C3a, C7a); 134.0 (C5, C6); 1_þ35.9 (C4⁰⁰);

149.5 (C6⁰⁰); 158.7 (C2⁰⁰); 168.5 (C1, C3). HRMS (ESI , MeOH):

(MþH)^þ, found m/z 299.0853. C₁₆H₁₅N₂O₂S requires 299.0854.

3.3.1. 2-(Pyridin-4⁰-ylsulfanyl)ethanamine (4-PSEA) (6b). General

procedure (B) was followed using 2-[2⁰-(pyridin-4⁰⁰-sulfanyl)ethyl]-

1H-isoindole-1,3(2H)-dione (3b) (2.20 g, 7.74 mmol) and hydrazine

hydrate (0.49 mL, 10.1 mmol) in dry EtOH (35 mL) for 16 h to give

the title compound 6b as an orange oil (0.99 g, 83%). n_max(neat):

3358s, 3286s, 3032s, 2926s, 2863m, 1928w, 1731w, 1659m, 1575s,

1538s, 1463s, 1407s, 1321m, 1220s, 1108s, 1066m, 985s, 866s, 804s,

3.2.3. 2-[3⁰-(Pyridin-4⁰⁰-ylsulfanyl)propyl]-1H-isoindole-1,3(2H)-di-

one (3d). General procedure (A) was modiﬁed as described by

Sohda et al.²⁶using N-(3-bromopropyl)phthalimide (6.63 g,

24.74 mmol), K₂CO₃(6.84 g, 49.47 mmol) and 4-mercaptopyridine

(2.75 g, 24.7 mmol) in DMF (50 mL) at ambient temperature for

3.5 h. The mixture was poured into water (100 mL) and the

resulting precipitate collected by vacuum ﬁltration and washed

with hexane. Recrystallisation from EtOAc afforded the product 3d

as pale pink crystals (3.57 g, 48%), mp 128e129 ^ꢀC (lit.²⁵mp

128e130 ^ꢀC). n_max(KBr): 3466w, 3027w, 2928w, 1780m, 1709s,

1578s, 1535w, 1457m, 1405s, 1377s, 1336m, 1220m, 1171w, 1113w,

1082m,1007s, 882m, 801m, 715s, 616w, 531m, 494m cm^ꢁ1. ¹H NMR

711s cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃):

. d 1.60 (br s, 2H, NH₂);

3.02e3.12 (m, 4H, H1, H2); 7.14 (d, J¼6.7 Hz, 2H, H3⁰, H5⁰); 8.39 (d,

J¼6.3 Hz, 2H, H2⁰, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d 35.0 (C2); 40.8

(C1); 121.2 (C3⁰, C5⁰); 148.7 (C4⁰); 149.5 (C2⁰, C6⁰). Mass spectrum

(ESI^þ): m/z 154.7 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 155.0640. C₇H₁₁N₂S requires 155.0643. The ¹H NMR data

were consistent with literature data.²⁶

2-(Pyridin-4⁰-ylsulfanyl)ethanamine (6b) was also prepared

following General procedure (C) as described in Section 3.4 using

4-chloropyridine hydrochloride (1.94 g, 12.9 mmol) to give

a brown oil, which was chromatographed (SiO₂, CH₂Cl₂/MeOH/

NH₄OH, 9:1:0.1) to afford the title compound 6b as a light brown

oil (1.73 g, 87%). The spectral data were consistent with those

given above.

(300 MHz, CDCl₃):

d

2.06 (m, 2H, H2⁰); 3.02 (t, J¼7.4 Hz, 2H, H3⁰);

3.86 (t, J¼6.8 Hz, 2H, H1⁰); 7.09 (dd, J¼4.6,1.6 Hz, 2H, H3⁰⁰, H5⁰⁰); 7.74

(m, 2H, H5, H6); 7.85 (m, 2H, H4, H7); 8.38 (dd, J¼4.6, 1.6 Hz, 2H,

H2⁰⁰, H6⁰⁰). ¹³C NMR. (75 MHz, CDCl₃):

d

27.9 (C2⁰); 28.4 (C3⁰); 37.1

(C1⁰); 121.0 (C3⁰⁰, C5⁰⁰); 123.6 (C4, C7); 132.2 (C3a, C7a); 134.3 (C5,

C6); 148.7 (C4⁰⁰); 149.6 (C2⁰⁰, C6⁰⁰); 168.5 (C1, C3). Mass spectrum

(ESI^þ): m/z 299.3 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 299.0855. C₁₆H₁₅N₂O₂S requires 299.0854.

3.3.2. 3-(Pyridin-2⁰-ylsulfanyl)propanamine (2-PSPA) (6c). General

procedure (B) was followed using 2-[3⁰-(pyridin-2⁰⁰-ylsulfanyl)-

propyl]-1H-isoindole-1,3(2H)-dione (3c) (3.00 g, 10.1 mmol) and

hydrazine hydrate (1.01 g, 20.1 mmol) in dry EtOH (40 mL) for 17 h

to give the title compound 6c as a yellow oil (1.63 g, 96%). n_max

(neat): 3362m, 3288m, 3044m, 2930s, 2860s, 1661m, 1577s, 1455s,

1415s, 1282m, 1125s, 1089w, 1044w, 986m, 882m, 824m, 758s,

3.3. General procedure (B) for hydrazinolysis of phthalimide

derivatives: preparation of 2-(pyridin-2⁰-ylsulfanyl)ethanamine

(2-PSEA) (6a)

The phthalimides 3aed were converted to the amines 6ae6d by

modifying the procedure outlined by Boschelli et al.²⁵as described

below for the preparation of 2-PSEA (6a).

724s, 619m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.53 (br s, 2H, NH₂);

1.85 (m, 2H, H2); 2.83 (t, J¼6.7 Hz, 2H, H1); 3.24 (t, J¼7.1 Hz, 2H,

H3); 6.95 (ddd, J¼7.3, 4.9, 1.1 Hz, 1H, H5⁰); 7.16 (dt, J¼8.1, 1.0 Hz, 1H,

H3⁰); 7.45 (ddd, J¼8.1, 7.3, 1.9 Hz, 1H, H4⁰); 8.41 (ddd, J¼4.9, 1.9,

2-[2’-(Pyridin-2⁰⁰-sulfanyl)ethyl]-1H-isoindole-1,3(2H)-dione (3a)

(1.50 g, 5.27 mmol) was suspended in dry EtOH (30 mL) to which

hydrazine hydrate (0.33 mL, 6.86 mmol) was added. The suspen-

sion was stirred for 16 h at ambient temperature. The mixture was

acidiﬁed (pH 1) with 2 M HCl and ﬁltered. The ﬁltrate was reduced

in vacuo and washed with EtOAc (2ꢂ40 mL). The aqueous solution

1.0 Hz, 1H, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d 27.3 (C3); 33.4 (C2);

40.9 (C1); 119.3 (C5⁰); 122.3 (C3⁰); 135.9 (C4⁰); 149.4 (C6⁰); 159.2

(C2⁰). Mass spectrum (ESI^þ): m/z 169.0 (MþH)^þ(100%). HRMS (ESI^þ,

MeOH): (MþH)^þ, found m/z 169.0793. C₈H₁₃N₂S requires 169.0799.

The ¹H NMR data were consistent with literature values.²⁶

480

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

3.3.3. 3-(Pyridin-4⁰-ylsulfanyl)propanamine (4-PSPA) (6d). General

procedure (B) was followed using 2-[3⁰-(pyridin-4⁰⁰-ylsulfanyl)-

propyl]-1H-isoindole-1,3(2H)-dione (3d) (2.50 g, 8.38 mmol) and

hydrazine hydrate (0.84 g, 16.8 mmol) in dry EtOH (50 mL) for 16 h

to give an orange oil (1.63 g). The crude oil was loaded onto a plug of

silica, washed (CH₂Cl₂/MeOH, 10:1) and the title compound 6d

eluted (CH₂Cl₂/MeOH/NH₃in MeOH, 10:1:0.2) as a yellow oil

(0.96 g, 68%). n_max(neat): 3358bs, 3032s, 2933s, 2861s, 1576s,

1538s, 1483s, 1436m, 1409s, 1321m, 1257w, 1221m, 1111m, 1066m,

3.4.2. 2-(4⁰-Methylpyridin-2⁰-ylsulfanyl)ethanamine (6f) (4-Me-2-

PSEA). 2-Aminoethanthiol hydrochloride (1.50 g, 13.2 mmol) was

added to a suspension of NaH (dry, 95%) (0.79 g, 32.9 mmol) in THF

(15 mL). The mixture was stirred for 10 min before the slow addi-

tion of HMPA (10 mL). 2-Chloro-4-methylpyridine (1.40 g,

11.0 mmol) was then added slowly and the mixture stirred at am-

bient temperature for 2 days. The reaction mixture was quenched

with water (10 mL) and the THF removed under reduced pressure.

The aqueous solution was extracted with CH₂Cl₂(2ꢂ10 mL). The

combined organic extract was washed with 1 M NaOH (10 mL),

dried (MgSO₄), ﬁltered and the solvent removed in vacuo to give

a brown oil (2.54 g). The crude product was puriﬁed by column

chromatography (SiO₂, CH₂Cl₂/MeOH/NH₄OH, 9:1:0.1) to yield the

title compound 6f as a light brown oil (0.70 g, 38%). n_max(neat):

3362m, 3287m, 3048w, 2922s, 2863m, 1594s, 1547s, 1467s, 1372s,

1284m, 1224m, 1122s, 1094s, 1070w, 1015w, 986m, 870s, 816s,

895m, 804s, 712s cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.62 (br s, 2H,

NH₂); 1.85 (m, 2H, H2); 2.86 (t, J¼6.8 Hz, 2H, H1); 3.05 (t, J¼7.2 Hz,

2H, H3); 7.12 (dd, J¼4.7, 1.6 Hz, 2H, H3⁰, H5⁰); 8.37 (dd, J¼4.7, 1.6 Hz,

2H, H2⁰, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d

28.2 (C3); 32.1 (C2); 41_þ.0

(C1); 120.8 (C3⁰, C5⁰); 149.3 (C2⁰, C6⁰); 149.3 (C4⁰). HRMS (ESI ,

MeOH): (MþH)^þ, found m/z 169.0799. C₈H₁₃N₂S requires 169.0799.

The ¹H NMR spectral data were consistent with literature values.²⁶

716m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.52 (br s, 2H, NH₂); 2.27

(s, 3H, CH₃); 2.99 (t, J¼6.4 Hz, 2H, H1); 3.28 (t, J¼6.3 Hz, 2H, H2);

3.4. General procedure (C) for reaction of 2-amino-

ethanethiol$HCl with halogen-substituted N-heterocycles

6.80 (d, J¼5.1 Hz, 1H, H5⁰); 7.03 (s, 1H, H3⁰); 8.26 (d, J¼5.1 Hz, 1H,

H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d 20.7 (CH₃); 33.0 (C2); 41.4 (C1);

120.9 (C5⁰); 122.9 (C3⁰); 147.1 (C4⁰); 148.9 (C6⁰); 158.0 (C2⁰). Mass

spectrum (ESI^þ): m/z 168.8 (MþH)^þ(100%). HRMS (ESI^þ, MeOH):

(MþH)^þ, found m/z 169.0794. C₈H₁₃N₂S requires 169.0799.

2-Aminoethanethiol hydrochloride (1.1e1.2 equiv) was sus-

pended in dry HMPA (1e4 mL). NaH (dry, 95%) (2.4e2.6 equiv) was

added portion-wise over 10e20 min while the reaction mixture

was cooled in an ice-cold water bath under a nitrogen atmosphere.

The mixture was stirred for 10 min at ambient temperature before

portion-wise addition of the desired halogenated heterocycle

(1.22e17.5 mmol, 1 equiv) over 10 min. The reaction mixture was

stirred at ambient temperature for 2 he2 days. Water was added

slowly to quench the reaction then the aqueous layer was extracted

with CH₂Cl₂, and the combined organic extract dried (MgSO₄), ﬁl-

tered and solvent removed in vacuo to give the crude product. TLC

on silica using CH₂Cl₂/MeOH/NH₃in MeOH, 9:0.8:0.2 showed the

products with R_fvalues in the range 0.15e0.20.

3.4.3. 2-(6⁰-Methylpyridin-2⁰-ylsulfanyl)ethanamine (6g) (6-Me-2-

PSEA). General procedure (C) was followed employing 2-chloro-6-

methylpyridine (1.50 g, 11.76 mmol). The crude brown oil was

puriﬁed on silica (EtOH) followed by (CH₂Cl₂/NH₃, 7 M in MeOH, 9:1)

to afford the title compound 6g as a brown oil (1.25 g, 63%). n_max

(neat): 3363m, 3286m, 3056w, 2924s, 2864m, 1566s, 1436s, 1373m,

1256w, 1161s, 1087m, 1035m, 1001m, 975m, 869s, 776s, 729m,

677m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.47 (br s, 2H, NH₂); 2.48 (s,

3H, CH₃); 2.99 (t, J¼6.4 Hz, 2H, H1); 3.28 (t, J¼6.4 Hz, 2H, H2); 6.82 (d,

J¼7.6 Hz, 1H, H3⁰or H5⁰); 7.00 (d, J¼7.6 Hz, 1H, H3⁰or H5⁰); 7.36

(apparent t, J¼7.6 Hz, 1H, H4⁰). ¹³C NMR (75 MHz, CDCl₃):

d 24.6

3.4.1. Preparation of 2-(pyridin-3⁰-ylsulfanyl)ethan-amine (6e) (3-

PSEA). General procedure (C) was followed employing 3-bromo-

pyridine (1.50 g, 9.49 mmol). Puriﬁcation of the crude product by

column chromatography (SiO₂, CH₂Cl₂/MeOH/NH₃in MeOH,

9:0.8:0.2) afforded the title compound 6e as an orange-brown oil

(0.73 g, 50%). n_max(neat): 3362m, 3288m, 3034w, 2923m, 2861w,

1595w, 1568m, 1468s, 1404s, 1322w, 1274w, 1229w, 1189w, 1109m,

(CH₃); 34.3 (C2); 42.0 (C1); 119.0, 119.4 (C3⁰, C5⁰); 136.4 (C4⁰); 157.6,

158.6 (C2⁰, C6⁰). Mass spectrum (ESI^þ): m/z 168.9 (MþH)^þ(100%).

HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 169.0794. C₈H₁₃N₂S re-

quires 169.0799.

3.4.4. 2-(2⁰-Phenylpyridin-4⁰-ylsulfanyl)ethanamine (6h) (2-Ph-4-

PSEA). General procedure (C) was followed employing 4-chloro-2-

phenylpyridine (0.90 g, 4.75 mmol). The crude orange-brown liquid

obtained was puriﬁed on silica (hexane/EtOAc/EtOH, 3:2:1) fol-

lowed by (CH₂Cl₂/MeOH/NH₃7 M in MeOH, 85:10:5) to afford the

title compound 6h as a yellow oil (0.90 g, 82%). n_max(neat): 3372bw,

3037w, 2924w, 1712w, 1663s, 1572s, 1535s, 1498w, 1464m, 1444m,

1381m, 1280w, 1244w, 1221w, 1179w, 1111m, 1074w, 986w, 804m,

1

1071w, 1019s, 862m, 796s, 706s cm^ꢁ1. H NMR (300 MHz, CDCl₃):

d

1.91 (br s, 2H, NH₂); 2.92 (m, 2H, H1); 3.03 (m, 2H, H2); 7.22 (ddd,

J¼8.0, 4.8, 0.8 Hz, 1H, H5⁰); 7.69 (ddd, J¼8.0, 2.4,1.6 Hz,1H, H4⁰); 8.43

(dd, J¼4.8, 1.6 Hz, 1H, H6⁰); 8.60 (dd, J¼2.4, 0.8 Hz, 1H, H2⁰). ¹³C NMR

(75 MHz, CDCl₃):

d

38.1 (C2); 40.9 (C1); 123.8 (C4⁰); 133.2 (C3⁰); 137._þ7

(C5⁰); 147.5, 150.8 (C2⁰, C6⁰). Mass spectrum (ESI): m/z 154.9 (MþH)

(100%). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 155.0642. C₇H₁₁N₂S

requires 155.0643. The corresponding 2HCl salt was prepared and

obtained as a white solid, mp 217.7 ^ꢀC (lit.⁵²mp 215 ^ꢀC).

774s, 733m, 695s cm^ꢁ1. ¹H NMR (400 MHz, CDCl₃):

d 1.42 (br s, 2H,

NH₂); 3.05 (m, 2H, H1); 3.14 (m, 2H, H2); 7.09 (dd, J¼5.3, 1.8 Hz, 1H,

H5⁰); 7.39e7.48 (m, 3H, H3⁰⁰, H4⁰⁰, H5⁰⁰); 7.57 (dd, J¼1.8, 0.7 Hz, 1H,

H3⁰); 7.94 (m, 2H, H2⁰⁰, H6⁰⁰); 8.48 (dd, J¼5.3, 0.7 Hz, 1H, H6⁰). ¹³C

The preparation of 6e was also carried out by modifying the

procedure described by Batmanghelich and Turner.³¹

NMR (100 MHz, CDCl₃): d

35.2 (C2); 41.0 (C1); 118.3, 119.5 (C3⁰, C5⁰);

A suspension of NaH dry, 95% (0.32 g, 13.5 mmol) and 2-amino-

ethanethiol hydrochloride (0.80 g, 10.3 mmol) in HMPA (3 mL) was

stirred in a cold water bath under a nitrogen atmosphere for 10 min

then allowed towarm toambient temperature. The reaction mixture

was cooled again and 3-bromopyridine N-oxide (1.50 g, 8.62 mmol)

was added. The mixture was stirred at ambient temperature for 3 h.

Phosphorus trichloride (1.5 mL) was added and the mixture stirred

for 20 h, with CHCl₃(5 mL) added once stirring became inhibited by

the thick slurry that formed. The reaction was poured into water

(10 mL), basiﬁed (pH 9) with 2 M NaOH solution and extracted with

CHCl₃(3ꢂ20 mL). The combined organic layer was dried (MgSO₄),

ﬁltered and solvent removed in vacuo and the resulting residue

puriﬁed as above to give 6e as an orange-brown oil (0.51 g, 38%).

127.2, 128.9 (C2⁰⁰, C3⁰⁰, C5⁰⁰, C6⁰⁰); 129.3 (C4⁰⁰); 139.3 (C1⁰⁰); 149.3

(C4⁰); 149.5 (C6⁰); 157.6 (C2⁰). Mass spectrum (ESI^þ): m/z 231.1

(MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 231.0961.

C₁₃H₁₅N₂S requires 231.0956.

3.4.5. 2-(6⁰-Phenylpyridin-2⁰-ylsulfanyl)ethanamine (6i) (6-Ph-2-

PSEA). General procedure (C) was followed employing 2-chloro-6-

phenylpyridine (1.80 g, 9.49 mmol). The crude liquid was puriﬁed

on silica (hexane/Et₂O/EtOH, 3:2:1) followed by (CH₂Cl₂/MeOH/

NH₃7 M in MeOH, 85:10:5) to afford the title compound 6i as

a yellow oil (1.79 g, 82%). n_max(neat): 3363bs, 3062s, 2926s, 2868m,

1959w, 1558s, 1496m, 1430s, 1387m, 1312m, 1239w, 1184m, 1159s,

1142s, 1089w, 1063m, 1025m, 982m, 907m, 803s, 758s, 694s cm^ꢁ1

.

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

481

¹H NMR (400 MHz, CDCl₃):

d

1.50 (br s, 2H, NH₂); 3.08 (t, J¼6.3 Hz,

nitropyridine (0.20 g, 1.26 mmol). The crude yellow liquid obtained

was dissolved in dry ether (3 mL) and dry MeOH (3 mL) to which

a saturated solution of HCl in ether (2 mL) was added. The resulting

precipitate was ﬁltered, washed with ice-cold dry ether and col-

lected as a yellow solid (132 mg). The yellow solid was dissolved in

water (5 mL), basiﬁed (pH 8e9) with saturated K₂CO₃solution and

extracted with CH₂Cl₂(3ꢂ5 mL). The combined organic extract was

dried (MgSO₄), ﬁltered and solvent removed in vacuo to yield 2-Cl-

4-PSEA as a brown oil (85 mg, 36%). n_max(neat): 3362bs, 2927s,

2868m, 2457w, 1569s, 1523s, 1456s, 1428m, 1371s, 1284m, 1230m,

2H, H1); 3.41 (t, J¼6.3 Hz, 2H, H2); 7.13 (dd, J¼7.8, 0.8 Hz, 1H, H3⁰);

7.40e7.48(m, 4H, H5⁰, H3⁰⁰, H4⁰⁰, H5⁰⁰); 7.53 (apparent t, J¼7.8 Hz,1H,

H4⁰); 8.01 (m, 2H, H2⁰⁰, H6⁰⁰). ¹³C NMR (75 MHz, CDCl₃):

d 34.2 (C2);

42.0 (C1); 116.0, 121.0 (C3⁰, C5⁰); 126.9, 128.9 (C2⁰⁰, C3⁰⁰, C5⁰⁰, C6⁰⁰);

129.2 (C4⁰⁰); 136.8 (C4⁰); 139.2 (C1⁰⁰); 156.9, 158.3 (C2⁰, C6⁰). Mass

spectrum (ESI^þ): m/z 231.3 (MþH)^þ(100%). HRMS (ESI^þ, MeOH):

(MþH)^þ, found m/z 231.0953. C₁₃H₁₅N₂S requires 231.0956.

3.4.6. 2-(6⁰-Methoxypyridin-2⁰-ylsulfanyl)ethanamine (6j) (6-OMe-2-

PSEA). General procedure (C) was followed employing 2-chloro-6-

methoxypyridine (1.50 g, 10.5 mmol). The crude brown liquid

obtained was dissolved in dry ether (3 mL) and dry MeOH (3 mL) and

a saturated solution of HCl in ether (3 mL) was added. The resulting

precipitate was collected by ﬁltration, washed with dry ether and

obtained as a beige solid (1.05 g). The solid was dissolved in water,

basiﬁed (pH 8e9) with a saturated K₂CO₃solution and extracted with

CH₂Cl₂(3ꢂ15 mL). The combined organic extract was dried (MgSO₄),

ﬁltered and solvent removed in vacuo to give the title compound 6j

as a brown oil (0.58 g, 30%). n_max(neat): 3372m, 2946m, 2862m,

1568s, 1462s, 1405s, 1347w, 1288s, 1260s, 1190m, 1147s, 1074m,

1151s, 1111m, 1082s, 986s, 795s, 750m cm^ꢁ^{1 1}H NMR (400 MHz,

.

CDCl₃):

d 1.38 (br s, 2H, NH₂); 3.02e3.11 (m, 4H, H1, H2); 7.05 (dd,

J¼5.4, 1.7 Hz, 1H, H5⁰); 7.15 (dd, J¼1.7, 0.4 Hz, 1H, H3⁰); 8.15 (dd,

J¼5.4, 0.4 Hz, 1H, H6⁰). ¹³C NMR (100 MHz, CDCl₃):

d 35.0 (C2); 40.7

(C1); 119.8, 120.5 (C3⁰, C5⁰); 149.0 (C6⁰); 151.9, 152.3 (C2⁰, C4⁰). Mass

spectrum (ESI^þ): m/z 190.8 (M (³⁷Cl)þH)^þ(36%), 188.8 (M (³⁵Cl)þ

H)^þ(100). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 189.0245.

C₇H₁₀ClN₂S requires 189.0253.

3.6. 2-(Quinolin-4⁰-ylsulfanyl)ethanamine (10a) (4-QSEA)

1025s, 984m, 884s, 785s, 729m cm^ꢁ1

.

¹H NMR (300 MHz, CDCl₃):

General procedure (C) was followed using 4-chloroquin-oline

(0.20 g, 1.22 mmol). The crude brown oil obtained was dissolved in

dry ether (3 mL) and dry MeOH (3 mL) and treated with a saturated

solution of HCl in ether (3 mL). The amine salt precipitated and was

collected by ﬁltration and washed with dry ether to give 10a$2HCl

as a white solid (240 mg, 71%), mp 221e224 ^ꢀC, (lit.⁵²mp

222e226 ^ꢀC). The solid was then dissolved in water, basiﬁed with

K₂CO₃and extracted with CH₂Cl₂(3ꢂ10 mL). The combined organic

extract was dried (MgSO₄), ﬁltered and solvent removed in vacuo to

give the title compound 10a as a brown oil (130 mg, 52%). n_max

(neat): 1563m, 1376w, 933m, 822w, 810w, 667w, 643m,

d

1.45 (br s, 2H, NH₂); 3.02 (t, J¼6.3 Hz, 2H, H1); 3.27 (t, J¼6.3 Hz, 2H,

H2); 3.93 (s, 3H, OCH₃); 6.42 (dd, J¼8.1, 0.7 Hz, 1H, H3⁰or H5⁰); 6.79

(dd, J¼7.5, 0.7 Hz, 1H, H3⁰or H5⁰); 7.38 (dd, J¼8.1, 7.5 Hz, 1H, H4⁰). ¹³C

NMR (75 MHz, CDCl₃): d

34.2 (C2); 41.8 (C1); 53.3 (OCH₃); 105.8 (C5⁰);

114.6 (C3⁰); 138.7 (C4⁰); 155.7 (C2⁰); 163.7 (C6⁰). Mass spectrum

(ESI^þ): m/z 185.0 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 185.0741. C₈H₁₃N₂OS requires 185.0749.

A similar reaction was undertaken in DMF at 60 ^ꢀC for 23 h and

gave 6j in 42% yield.

3.5. Preparation of 2-(4⁰-nitropyridin-2⁰-ylsulfanyl)-

ethanamine (6k) (4-NO₂-2-PSEA)

630m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.97 (br s, 2H, NH₂); 3.12

(m, 2H, H1); 3.24 (m, 2H, H2); 7.24 (d, J¼4.8 Hz, 1H, H3⁰); 7.56

(apparent ddd, J¼8.4, 6.9, 1.4 Hz, 1H, H6⁰or H7⁰); 7.72 (apparent

ddd, J¼8.4, 6.9, 1.4 Hz, 1H, H6⁰or H7⁰); 8.07 (ddd, J¼8.4, 1.4, 0.6 Hz,

1H, H5⁰or H8⁰); 8.17 (ddd, J¼8.4, 1.4, 0.6 Hz, 1H, H5⁰or H8⁰); 8.72 (d,

tert-Butyl (2-sulfanylethyl)carbamate (1.38 g, 7.81 mmol) was

dissolved in dry THF (20 mL) under a nitrogen atmosphere. NaH (dry,

95%) (0.24 g, 10.2 mmol) was added portion-wise over 45 min and

the mixture was then cooled to ꢁ78 ^ꢀC. A solution of 2-chloro-4-

nitropyridine N-oxide (1.50 g, 8.59 mmol) in dry THF (40 mL) was

added dropwise over 45 min to the cold carbamate solution. The

mixture was warmed to ambient temperature over 2 h, stirred for

a further 2 h and the THF was removed under reduced pressure. The

residue was treated with CHCl₃(50 mL) followed by dropwise ad-

dition of PCl₃(3 mL). The reaction mixture was stirred at ambient

temperature for 16 h and then poured slowly into water (50 mL). The

organic layer was separated and the aqueous layer washed with

CHCl₃(4ꢂ15 mL). The aqueous layer was basiﬁed (pH 8.5e10) with

2 M NaOH solution and extracted several times with CH₂Cl₂. The

combined organic extract was dried (MgSO₄), ﬁltered and solvent

removed in vacuo to give the title compound 6k as a red-brown oil

(ca. 95% pure) (1.06 g, ca. 68%). Preparative TLC (SiO₂, CH₂Cl₂/MeOH/

NH₃in MeOH, 9:0.8:0.2) using a sample of crude oil gave the pure

title compound 6k as a yellow oil. n_max(neat): 3372m, 3083m,

2927m, 2870m, 1600m, 1560s, 1531s, 1453m, 1427w, 1355s, 1234m,

1153s, 1100m, 1073w, 1015w, 982w, 910m, 881m, 838m, 761s, 734s,

J¼4.8 Hz, 1H, H2⁰). ¹³C NMR (75 MHz, CDCl₃):

d 35.4 (C2); 40.7 (C1);

116.5 (C3⁰); 123.8, 126.5, 129.9, 130.2 (C5⁰, C6⁰, C7⁰, C8⁰);_þ127.0 (C4a’);

147.1, 147.7 (C4⁰, C8a’); 149.4 (C2⁰). Mass spectrum (ESI ): m/z 205.0

(MþH)^þ(15%). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 205.0794.

C₁₁H₁₃N₂S requires 205.0799.

Reaction of 4-chloroquinoline (1.50 g, 9.17 mmol) with sodium

ethoxide (34.8 mmol) and 2-aminoethanethiol hydrochloride (1.56 g,

13.8 mmol) in dry ethanol (40 mL) at reﬂux for 21 h gave a 60:40

mixture of 10a and 4-ethoxyquinoline. Column chromatography on

silica (CH₂Cl₂/MeOH/NH₄OH, 9:2:0.2) gave 4-ethoxyquinoline⁵³as

a light brown oil (0.47 g, 30%). Trituration of a more polar fraction

gave N-[2-(quinolin-4-yl-sulfanyl)ethyl]quinolin-4-amine as a white

solid (0.32 g,11%), mp 217e219 ^ꢀC. ¹H NMR. (300 MHz, CDCl₃):

d 3.51

(t, J¼6.3 Hz, 2H, SCH₂); 3.77 (apparent q, J¼6.3 Hz, 2H, CH₂NH); 5.43

(br s, 1H, NH); 6.46 (d, J¼5.3 Hz, 1H, H3); 7.26 (d, J¼4.8 Hz, 1H, H3⁰⁰);

7.41 (apparent ddd, J¼8.3, 7.0, 1.3 Hz, 1H, ArH); 7.58 (apparent ddd,

J¼8.3, 7.0, 1.3 Hz, 1H, ArH); 7.64 (apparent ddd, J¼8.4, 7.0, 1.4 Hz, 1H,

ArH); 7.67 (m, 1H, ArH); 7.74 (apparent ddd, J¼8.4, 7.0, 1.4 Hz, 1H,

ArH); 8.00 (m, 1H, ArH); 8.10 (ddd, J¼8.4, 1.3, 0.6 Hz, 1H, ArH); 8.18

(ddd, J¼8.4, 1.4, 0.6 Hz, 1H, ArH); 8.58 (d, J¼5.3 Hz, 1H, H2) 8.69 (d,

679m cm^ꢁ1.¹H NMR (300 MHz, CDCl₃):

d 1.52 (br s, 2H, NH₂); 3.03 (t,

J¼6.4 Hz, 2H, H1); 3.35 (t, J¼6.4 Hz, 2H, H2); 7.66 (dd, J¼5.4, 2.0 Hz,

J¼4.8 Hz, 1H H2⁰⁰). ¹³C NMR (100 MHz, CDCl₃):

d 30.8 (SCH₂); 41.8

1H, H5⁰); 7.89 (dd, J¼2.0, 0.7 Hz,1H, H3⁰); 8.66 (dd, J¼5.4, 0.7 Hz,1H,

(CH₂NH); 99.2, 117.3 (ArCH); 119.0 (ArC); 119.4, 123.9, 125.2, 127.0

(ArCH); 127.1 (ArC); 129.5, 130.3, 130.4 (ArCH); 145.8, 148.0, 148.7,

149.1 (ArC); 149.4, 151.1 (ArCH). Mass spectrum (ESI): m/z 332.2

(MþH)^þ(100%). HRMS (ESI^þ, CH₂Cl₂:MeOH,1:4): (MþH)^þ, found m/z

332.1217. C₂₀H₁₈N₃S requires 332.1221.

H6⁰). ¹³C NMR (50 MHz, CDCl₃):

d

34.5 (C2); 41.6 (C1); 111.8 (C5⁰);

115.1 (C3⁰); 151.5 (C6⁰); 153.9,162.9 (C2⁰, C4⁰). Mass spectrum (ESI^þ):

m/z 200.0 (MþH)^þ(30%),183.0 (100). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 200.0494. C₇H₁₀N₃O₂S requires 200.0494.

3.5.1. 2-(2⁰-Chloropyridin-4⁰-yl-sulfanyl)ethanamine (2-Cl-4-PSEA).

3.6.1. 2-(Quinolin-2⁰-ylsulfanyl)ethanamine (10b) (2-QSEA). General

General procedure (C) was followed employing 2-chloro-4-

procedure (C) was followed employing 2-chloroquinoline (2.00 g,

482

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

12.2 mmol). The crude brown oil obtained was dissolved in dry

ether (5 mL) and treated with a saturated solution of HCl in ether

(3 mL). The resulting precipitate was collected by ﬁltration and

washed with ice-cold dry ether to give the 2HCl salt of 10b as

3.6.4. 2-(Pyrazin-2⁰-ylsulfanyl)ethanamine (10e) (2-PyzSEA). Gen-

eral procedure (C) was followed employing 2-chloropyrimidine

(2.00 g, 17.5 mmol). A portion of the crude material (4.41 g) was

puriﬁed on silica, (hexane:Et₂O:EtOH, 3:2:1) followed by (CH₂Cl₂/

MeOH/NH₃7 M in MeOH, 85:10:5) to give the title compound 10e

as a yellow oil (1.77 g, 85%). n_max(neat): 3358m, 3288m, 3058m,

2929m, 2865m, 1597m, 1562m, 1506s, 1460s, 1385s, 1285m, 1237w,

a

yellow solid (2.82 g), mp 210e211 ^ꢀC. Anal. Calcd for

C₁₁H₁₂N₂S$2HCl: C, 47.66; H, 5.09; N, 10.105. Found: C, 47.35; H,

5.19; N, 9.98%. The solid was dissolved in water, basiﬁed (pH 8e9)

with a saturated K₂CO₃solution and extracted with CH₂Cl₂

(3ꢂ15 mL). The combined organic extract was dried (MgSO₄), ﬁl-

tered and solvent removed in vacuo to give the title compound 10b

as a brown oil (2.10 g, 84%). n_max(neat): 3358m, 3281m, 3055m,

2925m, 2862m, 1614s, 1593s, 1555s, 1497s, 1452m, 1420s, 1376w,

1294s, 1138s, 1089s, 1017w, 942m, 861m, 817s, 780m, 750s cm^ꢁ1. ¹H

1181w, 1129s, 1072w, 1048s, 1006s, 835s, 760m cm^ꢁ^{1 1}H NMR

.

(400 MHz, CDCl₃):

d

1.54 (br s, 2H, NH₂); 3.02 (t, J¼6.4 Hz, 2H, H1);

3.30 (t, J¼6.4 Hz, 2H, H2); 8.21 (d, J¼2.7 Hz,1H, H6⁰); 8.34 (dd, J¼2.7,

1.6 Hz, 1H, H5⁰); 8.47 (d, J¼1.6 Hz, 1H, H3⁰). ¹³C NMR (100 MHz,

CDCl₃):

d

33.6 (C2); 41.6 (C1); _þ139.6 (C5⁰); 143.9, 144.3 (C3⁰, C6⁰);

156.8 (C2⁰). Mass spectrum (ESI ): m/z 156.1 (MþH)^þ(100%). HRMS

(ESI^þ, MeOH): (MþH)^þ, found m/z 156.0590. C₆H₁₀N₃S requires

156.0595. The corresponding 2HCl salt was prepared and obtained

as a white solid, mp 162e164 ^ꢀC (lit.³¹mp 161 ^ꢀC).

NMR (300 MHz, CDCl₃):

d

1.67 (br s, 2H, NH₂); 3.08 (bt, J¼6.2 Hz, 2H,

H1); 3.45 (t, J¼6.2 Hz, 2H, H2); 7.20 (d, J¼8.6 Hz, 1H, H3⁰); 7.40

(apparent ddd, J¼8.1, 6.9,1.2 Hz,1H, H6⁰or H7⁰); 7.61 (apparent ddd,

J¼8.4, 7.0, 1.5 Hz, 1H, H6⁰or H7⁰); 7.68 (apparent dd, J¼8.1, 1.5 Hz,

1H, H5⁰or H8⁰); 7.84 (dd, J¼8.7, 0.6 Hz, 1H, H4⁰); 7.91 (apparent dd,

3.6.5. 2-(2⁰,2⁰⁰:6⁰,2⁰⁰⁰-Terpyridin-4⁰-ylsulfanyl)-ethanamine (10f) (4-

TerPSEA). 2-Aminoethanethiol hydrochloride (0.76 g, 6.7 mmol) was

dissolved in dry DMF (6 mL) and NaH (dry, 95%) (0.38 g, 15.7 mmol)

was added portion-wise over 20 min while the reaction mixture was

cooled in an ice-cold water bath under a nitrogen atmosphere. The

mixture was stirred for 10 min at ambient temperature before por-

tion-wise addition of 4⁰-chloro-2,2⁰:6⁰,2⁰⁰-terpyridine³⁹(1.50 g,

8.6 mmol) over 10 min. The reaction mixture was heated at 50 ^ꢀC for

3 h. Water was added slowly to quench the reaction then the aqueous

layerwasextractedwithCH₂Cl₂(4ꢂ20mL)andthecombinedwashed

withsaturated NaCl solution (6ꢂ10 ml). The organic extract wasdried

(MgSO₄), ﬁltered and solvent removed in vacuo to give the product as

a light beige solid (1.64 g, 95%), mp 98e99 ^ꢀC (lit.³⁸mp 93.5e94.5 ^ꢀC).

J¼8.4, 1.2 Hz, 1H, H5⁰or H8⁰). ¹³C NMR (75 MHz, CDCl₃):

d 33.8 (C2);

41.9 (C1); 121.2 (C3⁰); 125.4 (ArCH); 126.1 (C4a’); 127.7, 128.1, 129.7

(ArCH); 135.5 (C4⁰); 148.4 (C8a’); 158.9 (C2⁰). Mass spectrum (ESI^þ):

m/z 205.1 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z

205.0802. C₁₁H₁₃N₂S requires 205.0799.

3.6.2. 2-(Isoquinolin-1⁰-ylsulfanyl)ethanamine (10c) (1-IQSEA). Gen-

eral procedure (C) was followed using 1-chloroisoquinoline (2.00 g,

12.2 mmol). The crude yellow liquid obtained was dissolved in dry

ether (6 mL) and dry MeOH (3 mL). A saturated solution of HCl in

ether (3 mL) was added, the resulting precipitate collected by ﬁl-

tration, washed with ice-cold dry ether and dried in vacuo to yield

the HCl salt of the title compound 10c as a white solid (3.12 g, 92%),

mp dec >120 ^ꢀC. Anal. Calcd for C₁₁H₁₂N₂S$2HCl$1.2H₂O: C, 44.2; H,

5.5; N, 9.4. Found: C, 44.2; H, 5.7; N, 9.3%. The white solid (1.50 g) was

dissolved in water, basiﬁed (pH 8e9) with saturated K₂CO₃solution

and extracted with CH₂Cl₂(3ꢂ15 mL). The combined organic extract

was dried (MgSO₄), ﬁltered and solvent removed in vacuo to yield

the title compound 10c as a yellow oil (1.09 g, 99%). n_max(neat):

3366m, 3293m, 3051m, 2926m, 2870m, 2806w,1766w,1619s,1589s,

1551s, 1494s, 1450m, 1402m, 1373m, 1337s, 1301s, 1261s, 1226m,

1201m, 1184m, 1150s, 1066m, 1025m, 989s, 909m, 866m, 816s, 745s,

¹H NMR (300 MHz, CDCl₃):

d

3.10 (t, J¼6.4 Hz, 2H, H1); 3.29 (t,

J¼6.4 Hz, 2H, H2); 7.33 (ddd, J¼7.5, 4.8, 1.2 Hz, 2H, H5⁰⁰, H5⁰⁰⁰); 7.85

(apparent td, J¼7.5, 1.8 Hz, 2H, H4⁰⁰, H4⁰⁰⁰); 8.37 (s, 2H, H3⁰, H5⁰); 8.60

(dt, J¼8.0,1.1 Hz, 2H, H3⁰⁰, H3⁰⁰⁰); 8.68 (ddd, J¼8.0, 4.8,1.0 Hz, 2H, H6⁰⁰,

H6⁰⁰⁰). ¹³C NMR (75 MHz, CDCl₃):

d 35.3 (C2); 41.1 (C1); 118.3 (ArCH);

121.6 (ArCH); 124.1 (ArCH); 137.1 (ArCH); 149.3 (ArCH); 150.9 (ArC);

155.35 (ArC); 156.1 (ArC). Mass spectrum (ESI^þ): m/z 309.1 (MþH)^þ

(100%). HRMS (ESI^þ, MeOH):(MþH)^þ, found: m/z309.1167. C₁₇H₁₇N₄S

requires 309.1174. The NMR data were consistent with literature

data.³⁸

675s, 648s cm^ꢁ1. ¹H NMR (400 MHz, CDCl₃):

d 1.43 (br s, 2H, NH₂);

3.05 (t, J¼6.4 Hz, 2H, H1); 3.47 (t, J¼6.4 Hz, 2H, H2); 7.29 (dd, J¼5.7,

0.6 Hz, 1H, H4⁰); 7.51 (apparent ddd, J¼8.4, 6.9, 1.4 Hz, 1H, H6⁰or

H7⁰); 7.61 (apparent ddd, J¼8.2, 6.9,1.2 Hz,1H, H6⁰or H7⁰); 7.70 (br d,

J¼8.2 Hz,1H, H5⁰or H8⁰); 8.20 (br d, J¼8.4 Hz,1H, H5⁰or H8⁰); 8.27 (d,

3.7. Preparation of 3-(pyridin-2⁰-yl)propanethiol (13a)

(2-MPP)

2-Picoline (3.62 g, 38.9 mmol) was dissolved in dry THF

(100 mL) containing TMEDA (0.5 mL). The solution was cooled to

ꢁ78 ^ꢀC under a nitrogen atmosphere before dropwise addition of

n-butyllithium, 0.88 M solution in hexanes, (66 mL, 58 mmol). The

solution was then allowed to warm to ꢁ10 ^ꢀC before being re-

cooled to ꢁ78 ^ꢀC. Thiirane (3.5 mL, 58 mmol) was added dropwise

to the reaction mixture. The solution was stirred for 30 min at

ꢁ78 ^ꢀC and then allowed to warm to ambient temperature. After

stirring overnight the reaction mixture was quenched with satu-

rated NH₄Cl (100 mL). THF was removed under reduced pressure

and the aqueous residue extracted with CH₂Cl₂(3ꢂ40 mL). The

combined organic extract was dried (Na₂SO₄), ﬁltered and solvent

removed under reduced pressure to give a brown oil (8.7 g). The

crude product was puriﬁed by distillation using a Kugelrohr ap-

paratus to afford the title compound 13a as a yellow oil (2.68 g,

45%), bp 150 ^ꢀC (oven)/0.5 mmHg. n_max(neat): 3376bm, 3065m,

3008m, 2929s, 2857s, 2533w, 1592s, 1567s, 1475s, 1434s, 1348w,

J¼5.7 Hz, 1H, H3⁰). ¹³C NMR (100 MHz, CDCl₃):

d 33.7 (C2); 41.8 (C1);

117.3 (C4⁰); 124.6, 127.0, 127.1 (ArCH); 127.2 (C8a’); 130.3 (ArCH);

135.5 (C4a’); 141.8 (C3⁰); 159.0 (C1⁰). Mass spectrum (ESI^þ): m/z

205.2 (MþH)^þ(100%), 188.2 (100). HRMS (ESI^þ, MeOH): (M^þ), found

m/z 204.0714. C₁₁H₁₂N₂S requires 204.0721.

3.6.3. 2-(Pyrimidin-2⁰-ylsulfanyl)ethanamine (10d) (2PymSEA).

General procedure (C) was followed employing 2-chloropyrimidine

(2.00 g, 17.5 mmol). The crude brown oil was puriﬁed by column

chromatography (SiO₂, CH₂Cl₂/MeOH/NH₄OH, 9:1:0.1) and the title

compound 10d was obtained as a yellow oil (1.90 g, 70%). n_max

(neat): 3362m, 3289m, 3030w, 2930m, 2866w, 1587s, 1564s, 1548s,

1459w, 1427m, 1380s, 1257w, 1202s, 1072w, 1016w, 870m, 803m,

774s, 748s, 630s cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.65 (br s, 2H,

NH₂); 3.03 (t, J¼6.4 Hz, 2H, H1); 3.26 (t, J¼6.4 Hz, 2H, H2); 6.96 (t,

J¼4.8 Hz, 1H, H5⁰); 8.50 (d, J¼4.8 Hz, 2H, H4⁰, H6⁰). ¹³C NMR

(75 MHz, CDCl₃):

d

35.0 (C2); 41.7 (C1); 116.7 (C5⁰); 157.4 (C4⁰, C6⁰);

1289m, 1150m, 1095w, 1050m, 994m, 751s cm^ꢁ1

.

¹H NMR

172.4 (C2⁰). Mass spectrum (ESI^þ): m/z 156.2 (MþH)^þ(75%), 139.2

(100). HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 156.0593. C₆H₁₀N₃S

requires 156.0595. The ¹H NMR spectral data were consistent with

literature values.²⁶

(300 MHz, CDCl₃):

d

1.40 (t, J¼7.9 Hz,1H, SH); 2.06 (m, 2H, H2); 2.57

(apparent q, J¼7.4 Hz, 2H, H1); 2.91 (t, J¼7.4 Hz, 2H, H3); 7.10 (ddd,

J¼7.5, 4.9, 1.2 Hz, 1H, H5⁰); 7.15 (d, J¼7.8 Hz, 1H, H3⁰); 7.59 (apparent

td, J¼7.7, 1.8 Hz, 1H, H4⁰); 8.52 (ddd, J¼4.9, 1.8, 0.9 Hz, 1H, H6⁰). ¹³C

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

483

NMR (75 MHz, CDCl₃):

d

24.3 (C1); 33.9, 36.9 (C2, C3); 121.3, 123.0

3.8. General procedure (D) for the preparation of PMSEA

derivatives

(C3⁰, C5⁰); 136.5 (C4⁰); 149.5 (C6⁰); 161.2 (C2⁰). Mass spectrum

(ESI^þ): m/z 305.4 (disulﬁdeþH)^þ(50%), 153.9 (MþH)^þ(100). HRMS

(ESI^þ, MeOH): (MþNa)^þ, found m/z 176.0510. C₈H₁₁NNaS requires

176.0510.

2-Aminoethanethiol hydrochloride (12.2e24.4 mmol) and the 2-,

3- or 4-(chloromethyl)pyridine hydrochloride (6.1e12.2 mmol) were

added to a solution of NaOH (24.4e48.8 mmol) in EtOH (20e40 mL)

with ice bath cooling. The reaction mixture was stirred for 30 min

before the ice bath was removed and the mixture then stirred at

ambienttemperature for2.5 h. The EtOHwasremovedunderreduced

pressure and water (25 mL) was added to the resulting residue. The

aqueous solution was extracted with CH₂Cl₂(3ꢂ25 mL) and the

combined organic layer was washed with brine (10 mL), dried

(K₂CO₃), ﬁltered and solvent removed in vacuo to afford a crude oil.

3.7.1. 3-(Pyridin-4⁰-yl)propanethiol (13b) (4-MPP). 4-Picoline (3.29 g,

35.3 mmol) was reacted with n-butyllithium, 0.88 M solution in

hexanes (60 mL, 53 mmol) and TMEDA (0.5 mL) in dry THF (100 mL)

as described for 13a (2-MPP) above. Dropwise addition at ꢁ78 ^ꢀC of

thiirane (3.2 mL, 53 mmol) to the reaction mixture resulted in

a colour change from red-orange to bright yellow. The solution was

stirred for 30 min at ꢁ78 ^ꢀC, allowed to warm to ambient temper-

ature and stirred overnight. Workup as described for 13a (2-MPP)

gave a brown oil (8.08 g). The crude oil was partially puriﬁed by

distillation using a Kugelrohr apparatus. The fractions contained

both the side product 2-butyl-4-methylpyridine⁵³as a yellow oil

(0.13 g, 2%) and crude 4-MPP, which was further puriﬁed by column

chromatography (SiO₂, EtOAc) to give 13b as a yellow oil (0.56 g,

10%), bp 150 ^ꢀC (oven)/0.5 mmHg (lit.⁴⁴84e85 ^ꢀC/0.25 mmHg). n_max

(neat): 3385bm, 3026m, 2932s, 1670m, 1602s, 1558m, 1496m, 1416s,

3.8.1. 2-(Pyridin-2⁰-ylmethylsulfanyl)ethanamine (15a) (2-PMSEA).

Reaction of 2-(chloromethyl)pyridine hydrochloride (1.00 g,

6.10 mmol), following the general procedure (D) gave a crude oil

(1.18 g). Column chromatography (SiO₂, CH₂Cl₂:MeOH:NH₄OH,

9:2:0.2) of the crude oil gave the title compound 15a as a yellow oil

(0.92 g, 89%). n_max(neat): 3356bs, 2922s, 1592s, 1568s, 1472s, 1435s,

1308w, 1214w, 1153w, 1088w, 1049m, 995m, 891m, 792m,

1295w, 1255w, 1220m, 1070w, 993m, 791m cm^ꢁ1

(300 MHz, CDCl₃):

.

¹H NMR

751s cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 1.75 (br s, 2H, NH₂); 2.61 (t,

d

1.38 (t, J¼7.9 Hz, 1H, SH); 1.95 (m, 2H, H2); 2.54

J¼6.3 Hz, 2H, H2); 2.85 (t, J¼6.3 Hz, 2H, H1); 3.84 (s, 2H, ArCH₂S);

7.17 (ddd, J¼7.5, 4.9,1.2 Hz,1H, H5⁰); 7.38 (apparent dt, J¼7.8, 0.9 Hz,

1H, H3⁰); 7.66 (apparent td, J¼7.7, 1.8, Hz, 1H, H4⁰); 8.52 (ddd, J¼4.9,

(apparent q, J¼7.3 Hz, 2H, H1); 2.74 (t, J¼7.6 Hz, 2H, H3); 7.11 (d,

J¼6.0 Hz, 2H, H3⁰, H5⁰); 8.49 (d, J¼6.0 Hz, 2H, H2⁰, H6⁰). ¹³C NMR

(75 MHz, CDCl₃):

d

24.0 (C1); 33.7, 34.4 (C2, C3); 124.0 (C3⁰, C5⁰);

1.8, 0.9 Hz, 1H, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d 34.7, 36.8, 39.9 (C1,

150.0 (C2⁰, C6⁰); 150.3 (C4⁰). Mass spectrum (ESI^þ): m/z 305.3

(disulﬁdeþH)^þ(100%), 153.9 (MþH)^þ(73).

C2, AreCH2eS); 121.0 (C5⁰); 12_þ2.1 (C3⁰); 135.8 (C4⁰)_þ; 148.2 (C6⁰);

157.9 (C2⁰). Mass spectrum (ESI ): m/z 168.8 (MþH) (100%). The

¹H NMR data of 15a were consistent with literature data.⁵⁵

4,4⁰-(Disulfanediyldipropane-3,1-diyl)dipyridine

was

also

obtained as an orange oil (0.48 g, 4%). n_max(neat): 3388bm, 3026m,

2931s, 2859m, 1602s, 1558m, 1496w, 1447m, 1415s, 1219m, 1069w,

3.8.2. 2-(Pyridin-3⁰-ylmethylsulfanyl)ethanamine (15b) (3-PMSEA).

Reaction of 3-(chloromethyl)pyridine hydrochloride (1.00 g,

6.10 mmol) following general procedure (D) gave a crude orange oil

(1.08 g). Column chromatography (SiO₂, CH₂Cl₂/MeOH/NH₄OH,

9:2:0.2) of the crude orange oil afforded the title compound 15b as

a yellow oil (0.77 g, 75%). n_max(neat): 3356bs, 2918s, 1591s, 1576s,

993m, 792m cm^ꢁ1. ¹H NMR (300 MHz, CDCl₃):

d 2.03 (m, 4H, CH₂);

2.66 (t, J¼7.1 Hz, 4H, CH₂); 2.72 (t, J¼7.6 Hz, 4H, CH₂); 7.11 (d,

J¼6.0 Hz, 4H, ArCH); 8.50 (d, J¼6.0 Hz, 4H, ArCH). ¹³C NMR (75 MHz,

CDCl₃):

d 29.5, 33.7, 37.8 (CH₂); 124.0 (ArCH); 149.9 (ArCH); 150.4

(ArC). Mass spectrum (ESI^þ): m/z 305.3 (MþH)^þ(100%). HRMS

(ESI^þ, MeOH): (MþH)^þ, found m/z 305.1140. C₁₆H₂₁N₂S₂requires

305.1146.

1478s, 1423s, 1253w, 1192m, 1100m, 1027s, 874m, 713s cm^ꢁ1

.

¹H

NMR (300 MHz, CDCl₃):

d

1.64 (br s, 2H, NH₂); 2.54 (t, J¼6.3 Hz, 2H,

H2); 2.85 (bt, J∼5.9 Hz, 2H, H1); 3.70 (s, 2H, ArCH₂S); 7.26 (ddd,

J¼7.9, 4.8, 0.8 Hz, 1H, H5⁰); 7.69 (m, 1H, H4⁰); 8.50 (apparent dd,

J¼4.8, 1.6 Hz, 1H, H6⁰); 8.53 (br d, J¼2.3 Hz, 1H, H2⁰). ¹³C NMR

3.7.2. 3-(Pyridin-3⁰-yl)propanethiol

(13c)

(3-MPP). 3-Picoline

(3.19 g, 34.3 mmol) was dissolved in dry THF (100 mL) containing

TMEDA (0.5 mL). Lithium diisopropylamide (LDA) was prepared by

dissolving diisopropylamine (6.2 mL, 44.5 mmol) in dry THF

(20 mL) under a nitrogen atmosphere, cooling to 0 ^ꢀC and adding n-

butyllithium, 0.88 M solution in hexanes (50 mL, 44.5 mmol)

dropwise. The LDA solution was stirred for a further 40 min at 0 ^ꢀC

and added dropwise to the picoline solution at ꢁ78 ^ꢀC under ni-

trogen. After complete addition (30 min) the solution was stirred

for a further 10 min at ꢁ78 ^ꢀC. The mixture was allowed to warm to

(75 MHz, CDCl₃):

d

33.3, 35.6, 40.9 (C1, C2, ArCH₂S); 123.7 (C5⁰);

134.3 (C3⁰); 136.5 (C4⁰); 148.7, 150.1 (C2⁰, C6⁰). Mass spectrum

(ESI^þ): m/z 168.8 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 169.0795, C₈H₁₃N₂S requires 169.0799.

3.8.3. 2-(Pyridin-4⁰-ylmethylsulfanyl)ethanamine (15c) (4-PMSEA).

Reaction of 4-(chloromethyl)pyridine hydrochloride (2.00 g,

12.20 mmol) following general procedure (D) gave a crude brown

oil (2.12 g). Column chromatography (SiO₂, CH₂Cl₂/MeOH/NH₄OH,

9:2:0.2) of the crude brown oil afforded the title compound 15c as

a yellow oil (1.93 g, 94%). n_max(neat): 3356bs, 2919s, 1601s, 1560m,

0

^ꢀC before re-cooling to ꢁ78 ^ꢀC. Thiirane (3.0 mL, 51 mmol) was

added dropwise to the reaction mixture. The solution was stirred

for a further 15 min at ꢁ78 ^ꢀC and then allowed to warm to ambient

temperature and stirred overnight. Workup as described for 13a

gave a brown oil (9.30 g). The crude product was partially puriﬁed

by distillation using a Kugelrohr apparatus. Column chromatogra-

phy (SiO₂, EtOAc) afforded the title compound 13c as a yellow oil

(0.15 g, 3%), bp 150 ^ꢀC (oven)/0.5 mmHg (lit.⁴⁰92e99 ^ꢀC/

0.15 mmHg). n_max(neat): 3384bw, 2929s, 2859m, 1664w, 1575m,

1478m, 1423s, 1299w, 1190w, 1103w, 1028m, 796m, 715s cm^ꢁ1. ¹H

1495w, 1415s, 1219w, 1068m, 994m, 882m, 819m cm^ꢁ1

.

¹H NMR

(300 MHz, CDCl₃):

d

1.75 (br s, 2H, NH₂); 2.53 (t, J¼6.3 Hz, 2H, H2);

2.84 (t, J¼6.3 Hz, 2H, H1); 3.67 (s, 2H, ArCH₂S); 7.26 (d, J¼6.0 Hz, 2H,

H3⁰, H5⁰); 8.54 (d, J¼6.0 Hz, 2H, H2⁰, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d

35.1, 35.8, 40.9 (C1, C2, ArCH₂S); 124.0 (C3⁰, C5⁰); 147.7 (C4⁰); 150.1

(C2⁰, C6⁰). Mass spectrum (ESI^þ): m/z 168.7 (MþH)^þ(100%). HRMS

(ESI^þ, MeOH): (MþH)^þ, found m/z 169.0796. C₈H₁₃N₂S requires

169.0799.

NMR (300 MHz, CDCl₃):

d

1.38 (t, J¼7.8 Hz, 1H, SH); 1.94 (m, 2H,

H2); 2.55 (m, 2H, H1); 2.75 (t, J¼7.6 Hz, 2H, H3); 7.22 (m, 1H, H5⁰);

7.51 (m, 1H, H4⁰); 8.44e8.46 (m, 2H, H2⁰, H6⁰). ¹³C NMR (75 MHz,

3.9. Preparation of 2-(2⁰-pyridin-2⁰⁰-ylethylsulfanyl)-

ethanamine (17a) (2-PESEA)

CDCl₃): d

23.9 (C1); 31.5, 35.1 (C2, C3); 123.5 (C5⁰); 136.1 (C4⁰); 136.7

(C3⁰); 147.6, 150.0 (C2⁰, C6⁰). Mass spectrum (ESI^þ): m/z 305.1

(disulﬁdeþH)^þ(100%), 153.6 (MþH)^þ(49). The ¹H NMR spectral

data of 13c were consistent with literature data.⁵⁴

2-(2⁰-Pyridin-2⁰⁰-ylethylsulfanyl)ethanamine (17a) was syn-

thesised according to the method described by Kaasjager et al.⁴⁴

484

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

2-Aminoethanethiol hydrochloride (2.16 g, 19.0 mmol) was

3.11. Ligand immobilisation

added to a solution of 2-vinylpyridine (2.00 g, 19.0 mmol) in dry

EtOH (50 mL). The mixture was stirred at reﬂux for 2 h before ad-

dition of 1 M NaOH in EtOH (20 mL) and reﬂux continued for

a further 30 min. The cloudy solution was ﬁltered to remove NaCl

and the ﬁltrate was concentrated in vacuo to give a brown oil

(3.66 g). The residue was dissolved in water (20 mL) and the

aqueous solution was extracted with CH₂Cl₂(3ꢂ20 mL). The com-

bined organic extract was dried (MgSO₄), ﬁltered and the solvent

removed under reduced pressure to give the title compound 17a as

a brown oil (3.30 g, 95%). n_max(neat): 3361s, 3286m, 3064m,

3008m, 2918s, 2856s, 1592s, 1568s, 1474s, 1436s, 1271m, 1211m,

Epichlorohydrin activated Sepharose 6 Fast Flow^Òwas prepared

by the method described by Jiang et al.³Sepharose 6 Fast Flow^Ò

(500 g) (Amersham Pharmacia Biotechnology, Uppsala, Sweden;

now GE Healthcare) was ﬁltered and washed extensively with

distilled water (5ꢂvolume of wet gel, 2.5 L). The gel was suction

dried and transferred to a Schott bottle. A 2 M NaOH solution

(500 mL) containing NaBH₄(0.937 g) (1.875 mg/mL) was added and

the resulting suspension mixed vigorously for 2 h on an Axyos or-

bital shaker. Epichlorohydrin (300 mL) was added and the gel slurry

was gently agitated for a further 6 days. The epoxy-activated resin

was collected by vacuum ﬁltration and washed with distilled water

(2.5 L). The activated resin was stored in a 20% (v/v) aqueous eth-

anol at 4 ^ꢀC until required for ligand immobilisation.

The ligands were attached to the activated Sepharose based on

the methods described by Jiang et al.³A 0.2 M solution of the ligand

was prepared by dissolving the heterocyclic compound in an ap-

propriate solvent (e.g., 25% or 50% (v/v) aqueous methanol). The

suction dried epichlorohydrin-activated resin was added to the

solution and the suspension mixed thoroughly on a rotating wheel

for 5 days at ambient temperature. The resulting adsorbent was

collected by vacuum ﬁltration, washed with the appropriate sol-

vent (ca. 10 bed volumes) followed by distilled water (ca. 10 bed

volumes). The adsorbent was stored in 20% (v/v) aqueous ethanol

solution at 4 ^ꢀC.

The extent of ligand immobilisation onto the epichlorohydrin-

activated Sepharose 6 Fast Flow^Òwas determined by the nitrogen

elemental analysis (Dairy Technical Services Ltd., Melbourne, Aus-

tralia). In brief, the adsorbent (approximately 10 g wet weight) was

collected by ﬁltration, washed with 25% (v/v) aqueous acetone

followed by 50% (v/v), then 75% (v/v), and ﬁnally 100% acetone (ca.

two bed volumes) and dried in vacuo to a constant weight. The

dried resin was accurately weighed and then analysed for total

nitrogen content to give the amount of immobilised ligand per

gram of dry resin. A typical standard deviation for replicates of

.

1151m, 1051m, 995m, 846bm, 754s cm^ꢁ^{1 1}H NMR (300 MHz,

CDCl₃):

d

1.41 (br s, 2H, NH₂); 2.62 (t, J¼6.5 Hz, 2H, H2); 2.87 (t,

J¼6.4 Hz, 2H, H1); 2.94 (m, 2H, H1⁰); 3.07 (m, 2H, H2⁰); 7.13 (ddd,

J¼7.5, 4.9, 1.2 Hz, 1H, H5⁰⁰); 7.18 (apparent dt, J¼7.8, 1.0 Hz, 1H, H3⁰⁰);

7.60 (apparent td, J¼7.7, 1.9 Hz, 1H, H4⁰⁰); 8.54 (ddd, J¼4.9, 1.8,

0.9 Hz, 1H, H6⁰⁰). ¹³C NMR (75 MHz, CDCl₃):

d

31.5 (C1⁰); 36.7 (C2);

38.7 (C2⁰); 41.3 (C1); 121.6 (C5⁰⁰); 123.3 (C3⁰⁰); 136.5 (C4⁰⁰); 149.5

(C6⁰⁰); 160.1 (C2⁰⁰). Mass spectrum (ESI): m/z 182.8 (MþH)^þ(100%).

The spectral data were consistent with literature data.⁴⁴

3.9.1. 2-(2⁰-Pyridin-4⁰⁰-ylethylsulfanyl)ethanamine (17b) (4-PESEA).

2-Aminoethanethiol hydrochloride (2.16 g, 19.0 mmol) was reacted

with 4-vinylpyridine (2.00 g, 19.0 mmol) in dry EtOH (50 mL) at

reﬂux for 2 h as described above for 17a to give a crude yellow oil

(3.18 g). The crude product was puriﬁed to remove remaining 4-

vinylpyridine using a plug of silica (EtOAc/hexane, 3:1, containing

10% EtOH). Elution (EtOAc/hexane, 3:1, containing 10% NH₃in

MeOH) to gave the desired compound 17b as a yellow oil (2.86 g,

82%). n_max(neat): 3359bs, 3070m, 2920s, 2858m, 1602s, 1559m,

1416s, 1321w, 1275w, 1222m, 1070w, 994m, 805s cm^ꢁ^{1 1}H NMR

.

(200 MHz, CDCl₃):

d 1.47 (br s, 2H, NH₂); 2.60e2.94 (m, 8H, H1, H2,

H1⁰, H2⁰); 7.13 (d, J¼6.0 Hz, 2H, H3⁰⁰, H5⁰⁰); 8.52 (d, J¼6.0 Hz, 2H, H2⁰⁰,

H6⁰⁰). ¹³C NMR (75 MHz, CDCl₃): 32.0 (C1⁰); 35.5, 36.4 (C2, C2⁰); 41.3

d

(C1); 123.8 (C3⁰⁰, C5⁰⁰); 149.1 (C4⁰⁰); 149.7 (C2⁰⁰, C6⁰⁰). Mass spectrum

(ESI^þ): m/z 182.9 (MþH)^þ(100%). HRMS (ESI^þ, MeOH): (MþH)^þ,

found m/z 183.0944. C₉H₁₅N₂S requires 183.0956.

these analyses was ꢃ14

mmol/g dry gel.

3.12. Static and dynamic adsorption studies

3.10. Preparation of 2-(pyridin-2⁰-ylsulfanyl)ethanol (19) (2-

PSEOH)

The static adsorption studies were carried out by incubating the

adsorbents with different concentrations of the puriﬁed mono-

clonal antibody (mAb) under a previously deﬁned buffer condition

2-Mercaptoethanol (1.09 g, 13.9 mmol) was dissolved in dry

HMPA (3 mL) to which NaH (0.40 g, 16.7 mmol) was added portion-

wise for 30 min under a nitrogen atmosphere. After complete ad-

dition, the mixture was stirred for 10 min before 2-bromopyridine

(2.00 g, 12.7 mmol) was added drop-wise. The mixture was stirred

at ambient temperature for 18 h and then at 40 ^ꢀC for 24 h. Water

(20 mL) was added slowly and the aqueous solution extracted with

CH₂Cl₂(3ꢂ15 mL). The combined organic extract was dried

(MgSO₄), ﬁltered and solvent removed in vacuo to give a yellow oil

(5.53 g). Column chromatography (SiO₂, ether/hexane, 4:1) gave

the title compound (19) as a yellow oil (ca. 95% pure) (1.54 g, ca.

72%). n_max(neat): 3362bs, 2929s, 2864s, 1580s, 1557s, 1455s, 1415s,

1283s,1232m,1149s,1045s,1016s, 942m, 878w, 758s, 724s cm^ꢁ1. ¹H

(pH 9.0, 600 mM Na₂SO₄). Each incubation (550

contained Tris buffer (55 L), mAb solution (110

slurry (20 L), 1 M Na₂SO₄(330 L) and H₂O (35 L). The mAb so-

lutions were prepared at the following concentrations: 330, 550,

770, 935, 1155, 1320, 1540 g/mL in 25 mM sodium acetate pH 5,

m

L total volume)

m

mL), adsorbent

m

220 mM sodium chloride buffer.

Following incubation, the free mAb concentrations in the su-

pernatants were measured in triplicate by the BCA protein assay.

The values of the maximum binding capacity (as mg mAb/mL ad-

sorbent) were determined from the derived isotherm using

established procedures. The BCA protein assays were carried out

according to the manufacturer’s speciﬁcations with the propriety

reagent A and reagent B (Pierce, Rockford, USA). An aliquot of the

supernatant from each incubation sample (20

triplicate in the wells of a 96-well microplate. A mixture of reagent

A (50 parts) and reagent B (1 part) was then prepared and 200 L of

the resulting solution added to each well. The plate was incubated

at 37 ^ꢀC for 30 min to allow the colour to develop. The absorbance

was then read on a Bio-Rad Microplate Reader Model 3550 at

595 nm and the protein concentration in the supernatant sample

determined from a standard curve using bovine serum albumin as

the control protein. The recoveries were determined from the ratio

of mass balances for the loaded and eluted mAb fractions, whilst

NMR (300 MHz, CDCl₃):

d

3.31 (t,¼J 5.5 Hz, 2H, H2); 3.93 (t,

J¼5.5 Hz, 2H, H1); 7.00 (ddd, J¼7.4, 5.0, 1.1 Hz, 1H, H5⁰); 7.25 (dt,

mL) was placed in

J¼8.1, 1.0 Hz, 1H, H3⁰); 7.48 (ddd, J¼8.0, 7.4, 1.9 Hz, 1H, H4⁰); 8.35

(ddd, J¼5.0, 1.8, 1.0 Hz, 1H, H6⁰). ¹³C NMR (75 MHz, CDCl₃):

d

33.9

m

(C2); 63.2 (C1); 119.9, 122.8 (C3⁰, C5⁰); 136.4 (C4⁰); 148.9 (C6⁰); 158.8

(C2⁰). Mass spectrum (ESI): m/z 155.8 (MþH)^þ(55%), 137.8 (100).

HRMS (ESI^þ, MeOH): (MþH)^þ, found m/z 156.0534. C₇H₁₀NOS re-

quires m/z 156.0483. The spectral data were consistent with liter-

ature data.⁴⁶

A similar reaction at ambient temperature for 25 h gave a 54%

conversion (¹H NMR spectroscopy) to the alcohol 19.

S.J. Mountford et al. / Tetrahedron 67 (2011) 471e485

485

the Q_maxvalues and the corresponding R²values for the static and

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Synthesis of N-heterocyclic ligands for use in affinity and mixed mode chromatography

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