Synthesis and evaluation of xanomeline analogs-Probing the wash-resistant phenomenon at the M1 muscarinic acetylcholine receptor

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Bioorganic & Medicinal Chemistry 16 (2008) 1376–1392

Synthesis and evaluation of xanomeline analogs—Probing

the wash-resistant phenomenon at the M₁muscarinic

acetylcholine receptor

Brian E. Kane,^bMarianne K. O. Grant,^aEsam E. El-Fakahany^aand David M. Ferguson^b,*

^aDepartment of Psychiatry, University of Minnesota, Minneapolis, MN 55455, USA

^bDepartment of Medicinal Chemistry, Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA

Received 16 July 2007; revised 15 October 2007; accepted 17 October 2007

Available online 22 October 2007

Abstract—A series of xanomeline analogs were synthesized and evaluated for binding at the M₁muscarinic acetylcholine receptor

(M₁receptor). Speciﬁcally, compounds that substitute the O-hexyl chain of xanomeline with polar, ionizable, or conformationally

restricted moieties were assessed for their ability to bind to the M₁receptor in a wash-resistant manner (persistent binding). From

our screen, several novel ligands that persistently bind to the M₁receptor with greater aﬃnity than xanomeline were discovered.

Results indicate that persistent binding may arise not only from hydrophobic interactions but also from ionic interactions with a

secondary M₁receptor binding site. Herein, a qualitative model that accounts for both binding scenarios is proposed and applied

to understand the structural basis to wash-resistant binding and long-acting eﬀects of xanomeline-based compounds.

1. Introduction

(Fig. 1), increased cognitive function of patients aﬀected

with AD (as measured by an assessment of memory, lan-

guage, and motor skills).⁴Despite these promising re-

sults, xanomeline was ultimately discontinued in Phase

III trials due to side eﬀects including salivation, nausea,

and diarrhea.⁵

Alzheimer’s disease (AD) is the most common cause of

dementia in the elderly, aﬄicting approximately 10%

of those over the age of 65, and 47% of those over age

84.¹With an aging population, the number of AD diag-

noses is expected to triple from 4.5 million Americans

diagnosed in 2000 to approximately 13 million by the

year 2050.²Considering these staggering statistics, there

is a tremendous need for therapeutics that can delay the

onset of AD or ameliorate its symptoms.

At approximately the same time that xanomeline was

discontinued, it was serendipitously discovered that it

binds to the M₁receptor in a wash-resistant manner.⁶

Speciﬁcally, preincubation of Chinese hamster ovary

(CHO) cells overexpressing the M₁receptor with

xanomeline followed by extensive washing resulted in

persistent receptor activation.^6,7This wash-resistant

activation, as measured by elevated basal levels of both

nNOS and phosphoinositide hydrolysis, can be silenced

by the addition of the non-selective muscarinic antago-

One of the many proposed therapeutic options to treat

AD is through the use of muscarinic receptor agonists.³

Based upon the observation that M₁receptors involved

in learning and memory are largely conserved in AD, it

has been speculated that pharmacologically selective M₁

receptor agonists would be viable therapeutics.³As

such, several M₁receptor agonists have been taken to

the clinic. One of these compounds, xanomeline

Keywords: Xanomeline; Alzheimer’s disease; Muscarinic; Plasma-

lemma diﬀusion microkinetic model; Long-acting; Drug design; M₁

receptor; Wash-resistant binding.

*

Corresponding author. Tel.: +1 612 626 2601; fax: +1 612 624

0139; e-mail: Ferguson@umn.edu

Figure 1. Structure of xanomeline.

doi:10.1016/j.bmc.2007.10.058

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1377

nist atropine.^6,7If atropine is washed oﬀ, reactivation

occurs. In order to further examine these unique proper-

ties, several other studies have been conducted.^7–12

Saturation binding experiments in cells preincubated

with xanomeline followed by washing oﬀ free ligand

exhibited reduced aﬃnity for [³H] N-methyl scopol-

amine (NMS) and a reduced receptor number.⁷As the

concentration of xanomeline was increased, the ob-

served eﬀects on both the K_dand B_maxof [³H]NMS

reached a limit.⁷Collectively, these data have led to

the assertion that xanomeline wash-resistant binding

occurs at an allosteric ‘exosite’ in a manner that is not

mutually exclusive with atropine.¹²

More recent studies have examined how modiﬁcation of

the hydrophobic tail of xanomeline aﬀects wash-resistant

binding.¹¹These studies modiﬁed the hexyl chain to short-

er alkyl groups in order to assess if non-speciﬁc hydropho-

bic interactions are the impetus for wash-resistant

binding. In summary, it was found that decreasing the

length of the hydrophobic chain to a propyl or methyl

group abolishes the wash-resistant binding.¹¹Although

these results point toward a hydrophobic site of interac-

tion, it is not clear if the interaction is in the receptor, on

the receptor surface, or in or on the membrane. Moreover,

the data is far from exhaustive and does not rule out the

existence of a pharmacophore speciﬁc exosite.

Scheme 1. Synthesis of alkyl derivatives.¹⁴Reagents and conditions:

(a) KCN, H₂O, AcOH, rt, 7.5 h; (b) NH₄Cl, H₂O/NH₄OH, rt, 18 h; (c)

S₂Cl₂, DMF, 0 ꢁC, 2h; (d) Na/ROH, 50 ꢁC, 3 h; (e) ROH, NaH, THF,

reﬂux, 18 h; (f) CH₃I, acetone, rt, 18 h; (g) NaBH₄, EtOH, 0 ꢁC, 1 h.

chloride in DMF aﬀorded 3-chloro-4-(3-pyridyl)-1,2,5-

thiadiazole (3), a key intermediate for the synthesis of

the majority of our derivatives.

The ethyl derivative of xanomeline was synthesized by

stirring 3 in a solution of sodium ethoxide.¹³The result-

ing pyridine derivative (4a) was subsequently converted

to its corresponding pyridinium salt via iodomethane in

acetone. Treatment with NaBH₄in ethanol (0 ꢁC for

1 h) aﬀorded 1,2,5,6-tetrahydropyridine 5a. Xanomeline

(5b), the hexyl derivative, was synthesized utilizing NaH

and 1-hexanol in THF.¹⁵Subsequent addition of inter-

mediate 3, followed by reﬂuxing for 18 h, led to 4b in

83% yield. Similar to 4a, this pyridine derivative was

methylated and reduced to aﬀord the 1,2,5,6-tetrahydro-

pyridine product (5b).

To date, there have been no studies that examine where

this potential binding site (speciﬁc or non-speciﬁc) may

be located. Studies involving reconstituted puriﬁed M₁

receptors implicate both the receptor and the membrane

lipids in xanomeline wash-resistant binding.¹¹In an ef-

fort to further deﬁne the structural requirements and po-

tential location of this binding site, we have synthesized

a series of xanomeline derivatives that replace the

hydrophobic O-hexyl chain with polar and ionizable

functional groups. In addition, we have synthesized sev-

eral molecules that have been shown to have functional

activity at the M₁receptor.¹³The ligands were subse-

quently tested for their ability to bind reversibly and

in a wash-resistant manner to the M₁receptor. Experi-

ments were also performed by varying the pH to evalu-

ate the eﬀect ionization has on both binding aﬃnity and

wash-resistant binding. The results are somewhat sur-

prising and suggest a correlation exists between ligand

structure and function that goes beyond simple hydro-

phobic eﬀects in mediating wash-resistant binding. The

data are further applied to develop a preliminary model

that accounts for these unique phenomena through the

existence of a secondary receptor exosite.

A series of hydroxyl derivatives were synthesized start-

ing from monoprotected diols (Scheme 2). Compound

2. Chemistry

Xanomeline and its analogs were synthesized largely

according to previously published procedures (Scheme

1).^13–15Sequential addition of potassium cyanide

and acetic acid to the commercially available 3-pyridine

carboxyaldehyde (1) aﬀorded 2-hydroxy-2-(3-pyri-

dyl)acetonitrile. Further reaction with ammonium

chloride in ammonium hydroxide led to 2-amino-2-(3-

pyridyl)acetonitrile (2). Reaction of 2 with sulfur mono-

Scheme 2. Synthesis of hydroxyl derivatives. Reagents and conditions:

(a) TBDPSCl, DMF/DIEA, rt, 12 h; (b) TBDPSCl, DIEA, CH₂Cl₂, rt,

2 h; (c) NaH, 3, THF, reﬂux, 18 h; (d) CH₃I, acetone, rt, 18 h; (e)

NaBH₄, EtOH, reﬂux, 12 h; (f) 1.0 M TBAF/THF, 0 ꢁC to rt, 3 h.

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B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

6a was monoprotected via dropwise addition of 1 equiv

of tert-butylchlorodiphenylsilane (TBDPSCl) into a

solution of ethylene glycol in DMF/DIEA.¹⁶The other

two diols, 1,3-propanediol and 1,4-butanediol, were

monoprotected according to a procedure reported by

Freeman et al.¹⁷In short, 2 equiv of the diol, 1 equiv

of DIEA, and 1 equiv of TBDPSCl were stirred in

CH₂Cl₂. In all cases, silica gel column chromatography

aﬀorded the monoprotected product. Similar to the

reaction that yielded 4b (NaH in THF), the monopro-

tected alcohols (7a–c) were reacted with intermediate 3

to aﬀord 8a–c. Again, the methyl pyridinium salt was

obtained by treatment with iodomethane in acetone.

Reduction with NaBH₄in EtOH at 0 ꢁC for 1 h did

not result in suﬃcient reduction of the pyridinium salt.

Therefore, reaction temperature and time were increased

until considerable amounts of 9a–c could be obtained.

Finally, deprotection of the TBDPS group with 1.0 M

tetrabutylammonium ﬂuoride (TBAF) in THF aﬀorded

compounds 10a–c.

reduced with NaBH₄to yield the desired Boc-amino

alcohols (12a–c).^19,20The other Boc-protected amino

alcohols (14a,14b) were synthesized from 3-aminopro-

an-1-ol and 5-amino-pentan-1-ol (13a and 13b). Treat-

ment with di-tert-butyl dicarbonate and Et₃N in

CH₂Cl₂aﬀorded compounds 14a and 14b in 90% and

99% yield, respectively.^21,22In addition to these primary

amines, a secondary amine, derived from N-methyl pyr-

rolidone (NMP), was synthesized. Acid mediated ring

opening,²³followed by Boc-protection,¹⁸yielded com-

pound 17. The carboxylic acid moiety was reduced to

the alcohol using BH₃THF.²⁴

Å

The Boc-protected amino alcohols were reacted with

intermediate 3 as described earlier. Speciﬁcally, deproto-

nation of the alcohol with 2 equiv of NaH in THF

followed by reﬂuxing with intermediate 3 for 18 h aﬀor-

ded compounds 18a–e and 21 (Scheme 4). Methylation

and reduction led to the 1,2,5,6-tetrahydropyridine

products (19a–e, 22). Deprotection of the Boc group

using 4.0 M HCl in dioxane or triﬂuoroacetic acid in

CH₂Cl₂aﬀorded the HCl or TFA salt, respectively.

Important to note in this series of reactions is that the

purity of 8a–c can be increased via a work-up procedure

consisting of an acid wash. Speciﬁcally, addition of a cit-

ric acid (pH = 4) solution to the CH₂Cl₂layer results in

intermediate 3 partitioning into the aqueous layer, leav-

ing 8a–c and the unreacted alcohol 7a–c largely in the

organic layer. Although this work-up procedure results

in slightly lower yields (due to minor amounts of 8a–c

lost in the acidic aqueous layer), it is deemed worthwhile

because it often eliminated the need for column

chromatography.

In the above sequence, it is noteworthy the reaction of 3

with 3-(tert-butoxycarbonylamino)-1-propanol (14b) led

to signiﬁcant amounts of undesired cyclic urethane.^22,25

In order to avoid this and increase the yield of 18b, it

was determined that the reaction mixture should be stir-

red at room temperature.

In addition to the above compounds, several analogs

that incorporate aromatic rings into the side chain were

synthesized (Scheme 5). Two such analogs, a 3-phenyl-2-

propyn-1-ol and a 4-phenyl-1-butanol derivative, have

been shown to possess signiﬁcant M₁receptor activity.¹³

Starting from the commercially available alcohols,

deprotonation with NaH in THF followed by addition

of intermediate 3 yielded products 24 and 25. Subse-

quent treatment with iodomethane and NaBH₄aﬀorded

compounds 26 and 27.

Amino derivatives were also synthesized in order to

determine what aﬀect, if any, a second positive charge

has in the wash-resistant binding of xanomeline. These

derivatives were synthesized from N-tert-butoxycar-

bonyl-protected amino alcohols, derived from commer-

cially available amino acids or amino alcohols (Scheme

3). Using a published method, glycine (11a), 4-aminobu-

tyric acid (GABA, 11b), and caproic acid (11c) were

Boc-protected.¹⁸Subsequent reaction with isopropyl-

chloroformate or ethylchloroformate aﬀorded a mixed

carboxylic-carbonic anhydride, which was subsequently

In order to assess the SAR of the phenyl ring of com-

pound 27, an imidazole and a pyrrole analog were

synthesized. Reaction of 10c with SOCl₂aﬀorded the

Scheme 3. Synthesis of Boc-amino alcohol derivatives. Reagents and conditions: (a) NaOH, dioxane/H₂O, Boc₂O; (b) alkyl chloroformate, THF, 0 ꢁC,

Å

30 min; (c) NaBH₄, MeOH; 0 ꢁC to rt, 30 min; (d) Et₃N/CH₂Cl₂/Boc₂O, 0 ꢁC to rt, 12 h; (e) concd HCl, reﬂux, 12 h; (f) BH₃THF, 0 ꢁC to rt, 12 h.

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1379

Scheme 4. Synthesis of amino derivatives. Reagents and conditions: (a) NaH, 3, THF, reﬂux, 18 h; (b) CH₃I, acetone, rt, 18 h; (c) NaBH₄, EtOH,

reﬂux, 12 h; (d) TFA/CH₂Cl₂, rt, 4 h; (e) 4.0 N HCl/dioxane, rt, 4 h.

Scheme 5. Synthesis of aromatic derivatives. Reagents and conditions: (a) ROH, NaH, THF, reﬂux, 18 h; (b) CH₃I, acetone, rt, 18 h; (c) NaBH₄,

EtOH, reﬂux, 3 h.

chloro-substituted derivative 28, which was reacted with

NaH and pyrrole or NaH and imidazole in DMF to af-

ford compounds 29 and 30, respectively (Scheme 6).

iodomethane and NaBH₄aﬀorded ethyl ester derivative

31. Glycolic acid derivatives were also synthesized.

Using similar reaction conditions as that for the hydro-

xyl and amino derivatives (NaH in THF), the glycolic

ethyl ester was formed. However, in the following reduc-

tion step, NaBH₄reduced the ester to primary alcohol

10a. Attempts with a milder reducing agent, NaCNBH₃,

The synthesis of several carboxylic acid derivatives was

pursued. Reaction of intermediate 3 with the sodium salt

of 5-hydroxypentanoic acid followed by treatment with

Scheme 6. Synthesis of imidazole and pyrrole derivatives. Reagents and conditions: (a) SOCl₂, CH₂Cl₂, 0 ꢁC to rt, 4 h; (b) pyrrole or imidazole, NaH,

DMF, reﬂux, 18 h.

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B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

Scheme 7. Synthesis of carboxylic acid derivatives. Reagents and conditions: (a) 5-hydroxypentanoic acid sodium salt, NaH, THF, reﬂux, 18 h; (b)

CH₃I, acetone, rt, 18 h; (c) NaBH₄, EtOH, reﬂux, 12 h; (d) glycolic acid ethyl ester, NaH, THF, reﬂux, 18 h; (e) LiOH 1:1 H₂O/ACN.

also resulted in the formation of 10a. Because of this, an

alternative synthetic route which involves initially form-

ing 1,2,5,6-tetrahydropyridine 32 was employed. Specif-

ically, 3 treated with iodomethane in acetone yielded the

corresponding pyridinium salt, which was subsequently

reacted with NaBH₄in ethanol. The resulting 1,2,5,6-

tetrahydropyridine compound (32) was reacted with gly-

colic acid ethyl ester and NaH in THF to yield ethyl es-

ter 33. Finally, saponiﬁcation with LiOH in 1:1 water/

acetonitrile aﬀorded compound 34 (Scheme 7).

how the structure of an analog is linked to its diﬀerential

ability to displace [³H]NMS in the two assays. Since a K_i

value for the wash-resistant binding studies cannot be

determined (due to non-competitive pseudo-irreversible

conditions), it is most ﬁtting to compare pIC₅₀values

(Àlog IC₅₀). In most cases (for the O-alkyl analogs

tested previously¹¹as well as the novel compounds de-

scribed herein), the diﬀerence between these two values

is approximately 1.75–2.05 orders of magnitude. Results

are summarized in Table 1.

Starting with the O-alkyl derivatives of xanomeline,

pIC₅₀values are in agreement with similar results ob-

tained in cell membranes.¹¹The ethyl derivative (5a),

which was not evaluated in our previous studies on the

O-alkyl derivatives, possesses a relatively high pIC₅₀va-

lue of 6.85 0.06. Despite this high pIC₅₀value, 5a did

not possess wash-resistant binding. This result is consis-

tent with our previous ﬁnding that a long alkyl chain is

necessary for such binding.¹¹

3. Results and discussion

Two receptor binding protocols were applied to assess

the physical behavior of the O-substituted xanomeline

analogs. The ﬁrst protocol, termed ‘continuous pres-

ence,’ is a traditional radioligand displacement assay

wherein cells are simultaneously incubated with a ﬁxed

concentration of radioligand and various concentrations

of the unlabeled analog. After 1 h, the cells are ﬁltered

and the amount of radioligand that remains bound is

measured. The other protocol, termed the ‘wash-resis-

tant paradigm,’ also measures how an unlabeled ligand

dose-dependently aﬀects the binding of a radioligand.

However, in these studies the radioligand is not added

until after the cells have been preincubated with the test

compound and extensively washed. This washing proce-

dure consists of diluting the incubation mixture with

ꢀ50 vol of buﬀer followed by centrifugation. The result-

ing pellet is subjected to this procedure (dilution and

centrifugation) an additional two times in order to en-

sure that only irreversible (or pseudo-irreversible) bind-

ing remains.⁷The cells are resuspended in buﬀer and the

radioligand [³H] N-methylscopolamine ([³H]NMS) is

added. Lastly, the incubation mixture is ﬁltered and

the residual bound radioactivity is measured. In all cases

non-speciﬁc binding was assessed in the presence of

10 lM atropine.

In addition to the O-alkyl derivatives, numerous polar

analogs were synthesized. These compounds were de-

signed in order to assess whether moieties that contain

the ability to hydrogen bond or form ionic interactions

would provide further insight into the mechanism

responsible for the wash-resistant binding. The

hydroxyl derivatives (10a–c) did not show a correlation

between chain length and pIC₅₀values (pIC₅₀values

in the continuous presence binding studies were in the

order of hydroxybutyl > hydroxyethyl > hydroxypro-

pyl; 6.51 0.04, 5.97 0.06, and 5.42 0.08, respec-

tively). Additionally, none of these derivatives possess

wash-resistant binding.

The amino derivatives also fail to show a correlation be-

tween the position of the polar group (the amine) and

the pIC₅₀values in the continuous presence experiments.

The pIC₅₀values of 20a–e (in order of increasing chain

length) were found to be 6.00 0.01, 6.79 0.10,

4.11 0.09, 5.72 0.13, and 6.19 0.09, respectively.

In contrast to their hydroxyl counterparts, several

A comparison of the results from these two diﬀerent

binding assays provides valuable information about

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1381

Table 1. Binding data for xanomeline analogs in continuous presence

and wash-resistant binding studies

amino derivatives were observed to possess wash-resis-

tant binding. The 6-aminohexyl derivative 20e possessed

excellent wash-resistance, displaying a pIC₅₀value of

5.40 0.07. In a descending order of their pIC₅₀values,

compounds 20b, 20d, and 20a possess wash-resistant

binding with pIC₅₀= 4.83 0.10, 4.35 0.19, and 3.96

ce:hsp sp="0.25"/>0.24, respectively. Interestingly, nei-

ther the 4-aminobutyl derivative (20d) nor its N-methyl

analog (23) was observed to bind in a wash-resistant

manner.

Compound

5a

R

pIC₅₀continuous pIC₅₀= washout

presence

conditions

6.85 0.06 (3)

N.B.

It was also of interest to determine if previously reported

xanomeline analogs possess wash-resistant binding. The

literature reveals that substitution of the hexyl chain

with alkylphenyl or alkynylphenyl moieties aﬀords com-

pounds that maintain potent M₁receptor activity.¹³

Since we had previously reported that alkyl derivatives

of butyl or larger possess wash-resistant binding,¹¹we

chose analogs that contained side chains of similar

length for synthesis and evaluation (26 and 27). Consis-

tent with previous reports, both 26 and 27 possess high

aﬃnity for the M₁receptor (pIC₅₀= 6.84 0.08 and

7.43 0.15 in the continuous binding assay, respec-

tively). Additionally, both ligands possess signiﬁcant

wash-resistant binding (pIC₅₀= 5.04 0.05 and

5.67 0.34, respectively). This is the ﬁrst reported result

of a xanomeline analog containing a phenyl ring that

binds in a wash-resistant manner. Presumably, the exo-

site does not discriminate against analogs that possess

moderate steric bulk.

LY1

LY2

LY3

5b

7.28 0.06 (4)

7.55 0.05 (4)

7.48 0.16 (3)

7.09 0.1 (3)

5.97 0.06 (2)

5.42 0.08 (2)

6.51 0.04 (2)

6.00 0.01 (4)

6.79 0.10 (4)

4.11 0.09 (4)

5.72 0.13 (3)

6.19 0.09 (4)

5.15 0.06 (5)

N.B.

4.11 0.2

5.07 0.2

5.31 0.04

N.B.

10a

10b

N.B.

10c

N.B.

20a

3.96 0.24

4.83 0.10

N.B.

20b

In addition to xanomeline derivatives containing a phe-

nyl ring, substitution of the hexyl chain with moieties

containing heteroaromatic rings has been reported.^26,27

Given our results that several derivatives with dual cat-

ionic character maintain wash-resistant binding, we syn-

thesized a potentially ionized imidazole derivative. In

the continuous binding assay, imidazole analog 29

possessed moderate aﬃnity for the M₁receptor

(pIC₅₀= 6.06 0.05). The aﬃnity of 29 was further

diminished in the wash-resistant binding assay

(pIC₅₀= 4.28 0.15). A comparison to the non-ioniz-

able pyrrole derivative 30 reveals similar activity in both

the continuous binding assay (pIC₅₀= 6.56 0.07) and

the wash-resistant assay (pIC₅₀= 4.54 0.26).

20c

20d

4.35 0.19

5.40 0.07

N.B.

20e

23

26

27

6.84 0.08 (3)

7.43 0.15 (4)

5.04 0.05

5.67 0.34

Ester derivative 31 was also tested. In the continuous

binding assay, it inhibited the binding of [³H]NMS to

a moderate extent (pIC₅₀= 7.23 0.06). In the wash-

resistant binding assay, it did not possess a residual

inhibitory eﬀect on [³H]NMS binding. Similar results

were also observed with carboxylic acid derivative 34.

In the continuous binding assay, a pIC₅₀value of

6.21 0.16 was obtained, and again, no residual wash-

resistant binding was observed.

29

30

31

34

6.06 0.05 (7)

6.56 0.07 (3)

7.23 0.06 (3)

6.21 0.16 (2)

4.28 0.15

4.54 0.26

N.B.

Upon further inspection of Table 1, amino derivatives

20c and 23 stand out because unlike the other alkyl-

amino derivatives, neither compound binds in a wash-

resistant manner. Since the positive charge of both of

these molecules is positioned at the same distance from

the thiadiazole ring, we hypothesized that the exosite

does not interact favorably with a positive charge at

The pIC₅₀values were determined in whole cell binding assays as

outlined in Section 5. The number of individual determination is

indicated in parentheses (n). Since the wash-resistant studies were done

in parallel (n) is the same for the wash-resistant binding studies. N.B.,

no binding.

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B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

Table 3. Diﬀerence between the pIC₅₀values in the two diﬀerent

binding protocols

the distance found for the butyl analogs. In order to

further explore this hypothesis, pH-dependent studies

with imidazole derivative 29 were conducted. It was

envisioned that the wash-resistant binding of 29 could

be abolished if the imidazole possessed a positive charge.

In order to protonate the imidazole, the buﬀer in the

binding studies was acidiﬁed to pH = 6.0. In the contin-

uous binding assay, 29 possessed a pIC₅₀value slightly

lower than the value obtained at pH = 7.4

(pIC₅₀= 5.64 0.10 at pH = 6.0 vs pIC₅₀= 6.06 0.05

at pH = 7.4, Table 2). Meanwhile, at pH = 8.0, 29 pos-

sessed the same pIC₅₀value as that observed under nor-

mal binding conditions (pIC₅₀= 6.06 0.05). This

suggests that the imidazole moiety is positively charged

at pH = 6.0 and neutral at pH = 7.4 and pH = 8.0. At

pH = 8.0, 29 maintained a similar pIC₅₀value as that

observed in Table 1 (4.27 0.41 vs 4.28 0.15).

Remarkably, at pH = 6.0, 29 lost the ability to bind in

a wash-resistant manner.

Compound pIC₅₀(CP) À pIC₅₀Compound pIC₅₀(CP) À pIC₅₀

(WO)

LY2

LY3

5b

3.44

2.41

1.78

2.04

1.96

1.37

20e

26

27

29

30

0.79

1.8

1.76

1.78

2.02

20a

20b

20d

CP, continuous presence; WO, washout.

ing protocol (compared to xanomeline) and an increased

pIC₅₀value (again compared to xanomeline) in the

wash-resistant binding studies. This is signiﬁcant be-

cause it suggests that ligand structure can modulate at

the site where the ligand interacts. If the chemical moie-

ties responsible for obtaining high wash-resistant bind-

ing can be delineated from those that do not, it may

be possible to rationally design ligands that bind wash-

resistantly.

In order to determine if the observed data were due to

pH-dependent changes in receptor structure, several

control experiments were conducted. Speciﬁcally, hexyl

derivative 5b (xanomeline) and pyrrole derivative 30

were tested at both pH = 6.0 and pH = 8.0. In both

cases, the binding proﬁles were consistent with previ-

ously determined values at pH = 7.4. Collectively, the

data suggest that topological changes in the receptor

are not responsible for the lack of wash-resistant bind-

ing of 29 (at pH = 6.0). Instead, it appears that the dif-

ference in wash-resistant binding is due to alterations of

the ionization state of the xanomeline analog.

Toward this end, it is of great importance to determine

how xanomeline elicits its wash-resistant binding. One

possible explanation is that it interacts at an allosteric

exosite found entirely within the M₁receptor. Such allo-

steric sites have been proposed for several other M₁

receptor agonists.^28,29According to Espinoza-Fonseca

and Trujillo-Ferrara,³⁰two of these allosteric sites are

located in the extracellular loops and the other is located

in the intracellular loops. Without chimeric or site-direc-

ted mutagenesis data, however, it is diﬃcult to assess

whether this hypothesis is well-founded. Wash-resistant

phenomena have also been rationalized by the sugges-

tion that some ligands preferentially partition non-spe-

ciﬁcally into the lipid bilayer. This type of interaction,

termed the plasmalemma diﬀusion microkinetic model,

does not fully explain the low aﬃnity of xanomeline

for several other neuronal receptors,^31,32nor does it ex-

plain the absence of wash-resistant xanomeline in cells

not transfected with the M₁receptor. A third hypothesis

that combines portions of the previous two is that

xanomeline binds at an interface of the receptor and

the cellular membrane. This hypothesis is supported

Also interesting to note from the data in Table 1 is that

some analogs display signiﬁcant variation in the diﬀer-

ence between the pIC₅₀values determined in the contin-

uous presence assay versus the pIC₅₀values determined

in the wash-resistant binding assay (Table 3). While

most of the compounds have a diﬀerence ranging from

1.75 to 2.05 log units, amino derivatives 20d and 20e fall

well below this range (1.37 and 0.79, respectively). The

relatively small diﬀerence between pIC₅₀values in the

two assays found for 20e is due to both a decreased

pIC₅₀value for the M₁receptor in the continuous bind-

Table 2. pH dependence on wash-resistant binding

Compound

R

pIC₅₀SEM continuous presence

pIC₅₀SEM washout conditions

5b

pH = 6: 7.74 0.13 (2)

pH = 8: 7.54 0.51 (2)

pH = 6: 5.62 0.43

pH = 8: 6.02 0.18

29

30

pH = 6: 5.64 0.10 (5)

pH = 8: 6.06 0.05 (5)

pH = 6: N.B.

pH = 8: 4.27 0.41

pH = 6: 6.39 0.08 (3)

pH = 8: 6.66 0.36 (3)

pH = 6: 4.40 0.33

pH = 8: 4.92 0.47

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1383

Receptor Exosite

OH

S

NH

S

N

O

N

O

N

O

N

O

H

P

O

P

N

S

N

NH₃

P

O

H

O

a

b

c

Figure 2. Schematic diagram of (a) benzthiazolone analog of salmeterol, (b) xanomeline (5b), and (c) 20e binding within the cellular membrane. In all

three models, the primary pharmacophore is also available for binding to the receptor.

by previous experiments that show that puriﬁed soluble

M₁receptors do not exhibit wash-resistant binding,

while puriﬁed receptors reconstituted into a liposome

do.¹¹Such a dual interaction has also been speculated

for the binding of salmeterol (a wash-resistant

D₂/b-adrenergic receptor agonist, Fig. 2a).³³Austin

et al. suggest that salmeterol partitions into the cellular

membrane in such a manner that the positive charge

interacts with the negatively charged polar head groups

while the hydrophobic region of the ligand interacts

with the hydrophobic chains contained within the mem-

brane (Fig. 2a).^34,35Although they do not propose direct

interactions of salmeterol with the D₂or the b-adrener-

gic receptor, it is reasonable to suggest that the exposed

benzthiazolone interacts with structural domains of the

receptor at the lipid–protein interface. It is reasonable

to propose an analogous mechanism for xanomeline

(Fig. 2b). Similar to the benzthiazolone, the thiadiazole

acts as a hydrogen bond acceptor for the positively

charged head group. Additional interactions are possi-

ble between the positively charged 1,2,5,6-tetrahydro-

pyridine ring and the negatively charged phosphate

moiety. The results also indicate that the addition of

charged groups to the lipophilic tail of xanomeline, such

as an amine (as demonstrated by 20e, Fig. 2c), increases

the wash-resistant binding aﬃnity. This lends consider-

able support to the general hypothesis that wash-resis-

tant binding is conferred through speciﬁc interactions,

perhaps at single or multiple exosites on the receptor.

study in which we reported that the hydrophobicity of

the O-alkyl group of xanomeline was strongly correlated

to its wash-resistant binding.¹¹The outcome of the cur-

rent study suggests that wash-resistant binding is not

simply related to hydrophobicity but may be a complex

phenomena involving one or more exosites on or around

the receptor. The data from Table 1 clearly show that

wash-resistant binding occurs for charged amino alkyl

substituted xanomeline analogs, while no continuous

binding is noted for the carboxyl derivative (34). It is

also signiﬁcant to note that a couple of the amino deriv-

atives possess pIC₅₀values in the wash-resistant binding

assay that are comparable to those of the pIC₅₀value in

the continuous presence assays. This lends strong

support to the general hypothesis that wash-resistant

binding is conferred through speciﬁc (as opposed to

non-speciﬁc) interactions with the receptor, or at the

lipid–receptor interface. While the results are provoca-

tive, it is important to note that wash-resistant binding

is also signiﬁcant for the O-arylalkyls reported here. It

is therefore diﬃcult to draw any strong conclusions at

this point in time as to the precise nature of this unique

physical property or to the structural architecture of the

secondary binding site.

In terms of therapeutics, the development of long-acting

drugs for the treatment of AD (and anti-psychotics)

would be highly advantageous, especially when consid-

ering patient compliance in treating mental health. The

ﬁnding that charged alkyl amino derivatives are active

is not trivial. This may be of particular importance since

past research has shown that the poor bioavailability

and a short plasma half-life (major detriments of the

development of xanomeline) are due to the O-butyl

carboxylic acid metabolite.³⁶Although the metabolic

pathways of these new compounds have yet to be stud-

ied, it is conceivable that this O-butyl carboxylic acid

4. Conclusions

In this paper, a series of xanomeline analogs were

synthesized and tested for their ability to bind in two

diﬀerent binding paradigms. These compounds were

speciﬁcally designed in order to complement a previous

1384

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

derivative can be avoided. It may therefore be of interest

to further evaluate the amino alkyls, as well as the phe-

nyl alkyls reported here for metabolic stability and tox-

icity, as these derivative may oﬀer new hope in the

design of long-acting AD therapeutics.

CH₂Cl₂(5· 100 mL). The combined organics were

washed with brine, dried over MgSO₄, and concentrated

to give 8.12 g (50% yield) of the title compound as a

brown oil. This brown oil was carried forward without

puriﬁcation. An analytical sample (>95% purity) was ob-

tained from silica gel column chromatography (4:1

1

EtOAc/MeOH). Overall yield from 1: 33%. H NMR

5. Experimental

5.1. Chemistry

(300 MHz, CDCl₃) d 8.82 (d, J = 2.4 Hz, 1 H), 8.67

(dd, J = 1.5, 4.8 Hz, 1H), 7.90–7.94 (m, 1H), 7.39 (dd,

J = 4.8, 7.9 Hz, 1H), 5.00 (t, J = 7.5 Hz, 1 H). HRMS

(ESI+) m/z: Calcd for C₇H₈N₃: 134.0713. Found:

134.0726.

¹H NMR spectra were recorded on a Varian Mercury

300 (300 MHz) spectrometer or

a

Varian 600

(600 MHz) spectrometer. Column chromatography

was conducted using silica gel 60 (40–63 lM). Yield

determinations were generally based on one experiment

and have not been optimized. Several of the pyridine

derivatives were not puriﬁed to >95% because in the fol-

lowing steps, the impurities are easily removed. In these

cases, the experimental yield is determined by calculat-

ing the fractional masses from the NMR spectrum of

the crude reaction mixture. High-resolution mass spec-

tra (HRMS) were collected from a TOF–ESI Agilent

LC–MS and analyzed using the Analyst QS software.

Reversed-phase high-performance liquid chromatogra-

phy (RP-HPLC) was performed on a Beckman Coulter

125S System Gold using an Agilent Zorbax Eclipse

XDB-C18 5.0 lM 4.6 · 150 mm column monitoring

UV at 210 nm on a 166 detector. The compounds tested

in the biological assays were all determined to be of

>95% purity based upon two separate chromatography

methods. Method A was a 20 min gradient method with

a ﬂow rate of 1.0 mL/min. Over the ﬁrst 15 min, the per-

centage of ACN/water was increased from 60% to 80%.

Over the ﬁnal 5 min, the gradient was increased to 100%

ACN. Method B was a gradient method that started

from a 65% MeOH/water with a ﬂow rate of 1.0 mL/

min. Here, the percentage of MeOH was increased to

100% over 20 min. Results were analyzed using the 32

Karat software package. Elemental analysis was not

performed.

5.1.2. 3-(3-Chloro-1,2,5-thiadiazol-4-yl)pyridine (3). Com-

mercially available (Aldrich) sulfur monochloride

(15.01 g, 111 mmol) was added to DMF (15 mL) at

0 ꢁC. To this, a solution of 2 (7.40 g, 55.6 mmol) in

DMF (7.5 mL) was added over an hour. After the addi-

tion was complete, the reaction mixture was stirred at

0 ꢁC for an additional 45 min, whereupon 30 mL of ice

water was added. The resulting precipitate was ﬁltered

and the ﬁltrate was chilled to 0 ꢁC. Over the next

5 min, 25 mL of a 9 M NaOH solution was added, result-

ing in a crystalline product (2.81 g). After ﬁltration, the

mother liquor was concentrated to yield a brown oil.

This oil was dissolved in CH₂Cl₂, washed with 5%

NaOH, brine, and concentrated. Silica gel column

chromatography (3:1 hexane/EtOAc) aﬀorded an

additional 3.51 g. In total, 6.58 g of a white crystalline

1

solid was obtained. Yield: 58%. H NMR (300 MHz,

CDCl₃) d 9.22 (dd, J = 0.9, 2.3 Hz, 1H), 8.73 (dd,

J = 1.6, 4.8 Hz, 1H), 8.28 (ddd, J = 1.7, 2.3, 8.0 Hz,

1H), 7.45 (ddd, J = 0.9, 4.9, 8.0 Hz, 1H). HRMS

(ESI+) m/z: Calcd for C₇H₅N₃SCl: 197.9887. Found:

197.9896.

5.1.3. 3-(3-(Ethoxy)-1,2,5-thiadiazol-4-yl)pyridine (4a).

Elemental sodium (0.20 g, 8.7 mol) was slowly added

to ethanol (10 mL). After the sodium had completely

dissolved, compound 3 (294 mg, 1.49 mmol) in ethanol

(5 mL) was added. The reaction was stirred for 3 h at

50 ꢁC and then evaporated. The resulting residue was

dissolved in H₂O and extracted with CH₂Cl₂

(3· 50 mL). The combined organics were washed with

brine, dried over MgSO₄, and concentrated to yield

254 mg of a white solid. Yield: 82%. ¹H NMR

(300 MHz, CDCl₃) d 9.42 (d, J = 0.8 Hz, 1H), 8.66

(dd, J = 1.8, 4.7 Hz, 1H), 8.45 (ddd, J = 1.7, 2.3,

8.0 Hz, 1H), 7.40 (ddd, J = 0.8, 4.8, 7.8 Hz, 1H), 4.51

(q, J = 6.2 Hz, 2H), 1.46 (t, J = 6.6 Hz, 3H). HRMS

(ESI+) m/z: Calcd for C₉H₁₀N₃SO: 208.0539. Found:

208.0536.

5.1.1. 2-Amino-2-(3-pyridyl)acetonitrile (2). Commercially

available 3-pyridinecarboxaldehyde (22.09 g, 0.206 mol)

was added dropwise (over a 60 min period) to a stirred

solution of potassium cyanide (14.77 g, 0.277 mol)

in H₂O (62 mL) at 5 ꢁC. Once addition was complete,

12.8 mL of glacial acetic acid was added over a 30 min

period (5 ꢁC). The reaction was stirred at room tempera-

ture for 6 h and then cooled to 5 ꢁC, in order to promote

precipitation. However, since the product failed to precip-

itate, the aqueous solution was extracted with EtOAc (4·

200 mL). The combined organic extracts were washed

with H₂O (200 mL) and brine (200 mL) and dried over

MgSO₄. The solvent was concentrated to yield 18.01 g

(66%) of the crude cyanohydrin product as a brown oil.

¹H NMR (300 MHz, CDCl₃) d 8.58 (d, J = 2.4 Hz,

1H), 8.51 (dd, J = 1.5, 4.8 Hz, 1 H), 7.93 (dt, J = 1.8,

7.8 Hz, 1H), 7.40 (dd, J = 4.5, 7.8 Hz, 1 H). The crude

cyanohydrin (16.42 g, 122 mmol) was added to a solution

of NH₄Cl (40.10 g, 750 mmol) in H₂O (100 mL) and 29%

NH₄OH (10 mL). The reaction mixture was stirred at

room temperature for 18 h and extracted with

5.1.4. 3-(3-(Hexyloxy)-1,2,5-thiadiazol-4-yl)pyridine (4b).

A suspension of sodium hydride (447 mg, 11.2 mmol,

60% dispersion in mineral oil) was added to a solution

of 1-hexanol (570 mg, 5.58 mmol) in THF (20 mL) at

0 ꢁC. This reaction mixture was reﬂuxed for 1 h and then

cooled to 0 ꢁC. After cooling, intermediate 3 (440 mg,

2.23 mmol) in THF (5 mL) was added. The reaction

mixture was reﬂuxed for 8 h, cooled, and then concen-

trated. The residue was taken up in CH₂Cl₂, washed

with 5% NaOH, brine, and dried over MgSO₄. The or-

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1385

ganic layer was concentrated to yield crude 4b as a white

solid (490 mg). Yield: 83%. ¹H NMR (300 MHz,

CDCl₃) d 9.41 (d, J = 1.5 Hz, 1H), 8.66 (dd, J = 1.7,

4.8 Hz, 1H), 8.44 (dt, J = 1.8, 7.8 Hz, 1H), 7.40 (ddd,

J = 0.7, 4.8, 8.0 Hz, 1H), 4.53 (t, J = 6.6 Hz, 2H),

1.85–1.95 (m, 2H) 1.45–1.62 (m, 2H), 1.32–1.55 (m,

4H), 0.87–0.91 (m, 3H). HRMS (ESI+) m/z: Calcd for

C₁₃H₁₈N₃SO: 264.1165. Found: 264.1187.

and concentrated in vacuo. The residue was puriﬁed

by silica gel column chromatography eluting with hex-

ane/acetone (3:1) to furnish 7a (368 mg) as a white solid.

1

Yield: 76%. H NMR (300 MHz, CDCl₃) d 7.70–7.74

(m, 4H), 7.39–7.49 (m, 6H), 3.68–3.82 (m, 4H), 2.34 (t,

J = 6.2 Hz, 1H) 1.05 (s, 9H). HRMS (APCI+) m/z:

Calcd for C₁₉H₂₇O₂Si: 301.1618. Found: 301.1602.

5.1.5.4. 3-tert-Butyldiphenylsilyloxy-1-propanol (7b).

To a solution of 1,3-propanediol (4.19 g, 55.0 mmol)

and DIEA (10 mL) in CH₂Cl₂(20 mL) was added

TBDPSCl (1.62 g, 5.92 mmol) in CH₂Cl₂(10 mL) drop-

wise. The solution was stirred at room temperature for

12 h and then concentrated. Column chromatography

(3:1 hexane/EtOAc) of the crude residue aﬀorded a

5.1.5. General procedure for the synthesis of 1,2,5-thia-

diazol-4-yl 1,2,5,6-tetrahydro-1-methylpyridines: method

A

5.1.5.1. 3-(3-(Ethoxy)-1,2,5-thiadiazol-4-yl)-1,2,5,6-

tetrahydro-1-methylpyridine (5a). To a stirred solution

of 4a (252 mg, 1.22 mmol) in acetone (ꢀ5 mL) was

added iodomethane (346 mg, 2.44 mmol). The reaction

mixture was stirred at room temperature for 18 h. The

pyridinium salt precipitated from solution and was

ﬁltered and washed with cold acetone. To a stirred

solution of the pyridinium salt in absolute ethanol

(296 mg, 0.85 mmol) at 0 ꢁC was added NaBH₄

(136 mg, 3.4 mmol). The mixture was reﬂuxed for 3 h,

concentrated, and partitioned between CH₂Cl₂and

water. The aqueous layer was separated and further ex-

tracted with CH₂Cl₂(2· 50 mL). The combined organics

were washed with brine, dried over Na₂SO₄, and con-

centrated to furnish a brown oil. Silica gel column chro-

matography eluting with EtOAc/MeOH (3:1) aﬀorded

5a as a brown oil (92 mg). Yield: 33%. ¹H NMR

(300 MHz, CDCl₃) d 7.04 (m, 1H), 4.46 (q, J = 7.1 Hz,

2H), 3.42 (d, J = 1.7 Hz, 2H), 2.54 (t, J = 5.6 Hz, 2H),

2.32–2.49 (m, 5H), 1.43 (t, J = 7.1 Hz, 3H). HRMS

(ESI+) m/z: Calcd for C₁₀H₁₆N₃SO: 226.1009. Found:

226.1019. It is important to note that in many of the

methylation reactions there was little or no precipita-

tion. In these cases, the reaction mixture was concen-

trated, Et₂O was added, and then concentrated in

vacuo. Although this sometimes provided a solid (which

was subsequently washed with cold acetone), much of

the time an oil resulted. In all cases, the mixture contain-

ing the pyridinium salt was carried forward without fur-

ther puriﬁcation.

1

white solid (5.01 g). Yield 88%. H NMR (300 MHz,

CDCl₃) d 7.70–7.74 (m, 4H), 7.39–7.49 (m, 6H), 3.68–

3.82 (m, 4H), 2.34 (t, J = 6.2 Hz, 1H) 1.05 (s, 9H).

HRMS (APCI+) m/z: Calcd for C₂₀H₂₉O₂Si: 315.1775.

Found: 315.1506.

5.1.5.5. 4-tert-Butyldiphenylsilyloxy-1-butanol (7c).

The title compound was obtained using the same meth-

od as described for 7b. The resulting residue was puriﬁed

by column chromatography eluting with hexane/EtOAc

1

(3:1). Yield 81%. H NMR (300 MHz, CDCl₃) d 7.66–

7.69 (m, 4H), 7.36–7.46 (m, 6H), 3.82–3.87 (m, 4H),

2.40 (t, J = 5.5 Hz, 1H) 1.80 (p, 4H) 1.05 (s, 9H). HRMS

(APCI+) m/z: Calcd for C₂₀H₂₉O₂Si: 329.1931. Found:

329.1896.

5.1.6. General procedure for the synthesis of derivatives

8a–8c: method B

5.1.6.1. 3-(3-((2-(tert-Butyldiphenylsilyloxy)ethyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine(8a). Compound 7a (540 mg,

1.59 mmol) was dissolved in THF (ꢀ15 mL) and chilled

to 0 ꢁC. Sodium hydride (127 mg, 3.18 mmol, 60% disper-

sion in mineral oil) was added, and the reaction mixture

was reﬂuxed for 1 h. The solution was chilled to 0 ꢁC,

and intermediate 3 (470 mg, 2.38 mmol) in THF

(10 mL/mmol) was added. The reaction mixture was

reﬂuxed for 18 h, cooled, and then concentrated. The

residue was dissolved in CH₂Cl₂(50 mL), washed with

aqueous citric acid, pH = 4 (3· 50 mL), 5% NaOH, brine,

dried over MgSO₄, and concentrated to aﬀord crude 8a

(609 mg) as a colorless oil. Yield: 83%. ¹H NMR

(300 MHz, CDCl₃) d 9.38 (d, J = 1.9 Hz, 1H), 8.59 (dd,

J = 1.7, 4.8 Hz), 8.49 (dt, J = 1.8, 7.2 Hz, 1H), 7.64–7.79

(m, 5H), 7.29–7.43 (m, 6H), 4.67 (t, J = 4.9 Hz, 2H),

4.08 (t, J = 4.9 Hz, 2H), 1.12 (s, 9H). HRMS (ESI+)

m/z: Calcd for C₂₅H₂₈N₃O₂SSi: 462.1666. Found:

462.1653.

5.1.5.2. 3-(3-(Hexyloxy)-1,2,5-thiadiazol-4-yl)-1,2,5,6-

tetrahydro-1-methylpyridine (5b) HCl salt. The title com-

pound was synthesized from 4b according to method A.

The freebase was converted to its hydrochloride salt by

bubbling HCl (g) through an ethereal solution. Yield:

40%. ¹H NMR (300 MHz, CDCl₃) d 7.05–7.09 (m,

1H), 4.44 (t, J = 6.6 Hz, 2H), 3.52–3.55 (m, 2H), 2.67

(t, J = 6.0 Hz, 2H), 2.44–2.49 (m, 5H), 1.77–1.87 (m,

2H), 1.30–1.46 (m, 6H), 0.91 (t, J = 6.0 Hz, 3H). HRMS

(ESI+) m/z: Calcd for C₁₄H₂₄N₃SO: 282.1635. Found:

282.1651.

5.1.6.2. 3-(3-((3-(tert-Butyldiphenylsilyloxy)propyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (8b). The title compound

was synthesized according to method B. Yield: 75%.

¹H NMR (300 MHz, CDCl₃) d 9.29 (dd, J = 0.9,

2.3 Hz, 1H), 8.60 (dd, J = 1.7, 4.8 Hz, 1H), 8.30 (ddd,

J = 1.7, 2.3, 8.0 Hz, 1H), 7.58–7.78 (m, 5H), 7.24–7.40

(m, 6H), 4.66 (t, J = 6.0 Hz, 2H), 3.87 (t, J = 6.0 Hz,

2H), 2.11 (p, J = 6.0 Hz, 2H), 1.12 (s, 9H). HRMS

(ESI+) m/z: Calcd for C₂₆H₃₀N₃O₂SSi: 476.1823.

Found: 476.1887.

5.1.5.3. 2-tert-Butyldiphenylsilyloxy-1-ethanol (7a).

a mixture containing ethylene glycol (1.00 g,

To

16.1 mmol), DMF (48 mL), and DIEA (21 mL) was

added TBDPSCl (4.65 g, 16.92 mmol) dropwise. The

reaction mixture was stirred for 12 h and was then

quenched with cold water (ꢀ200 mL), and extracted

with Et₂O. The organic layer was separated, successively

washed with 1 M HCl, saturated NaHCO₃, and brine,

1386

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

5.1.6.3. 3-(3-((4-(tert-Butyldiphenylsilyloxy)butyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (8c). The title compound

was synthesized from according to method B. Yield:

method C. However, after the THF solution was con-

centrated, the resultant residue was partitioned between

Et₂O and 5% NaOH. The organic layer was separated,

washed with brine, dried over Na₂SO₄, and concen-

trated. Column chromatography eluting with CH₂Cl₂/

MeOH/Et₃N (15:1:0.2) aﬀorded alcohol 10a. Yield:

20%. ¹H NMR (300 MHz, CDCl₃) d 7.04 (m, 1H),

4.57 (t, J = 4.5 Hz, 2H), 3.99 (t, J = 4.5 Hz, 2H), 3.45–

3.49 (m, 2H), 2.61 (t, J = 5.7 Hz, 2H), 2.44–2.50 (m,

5H). HRMS (ESI+) m/z: Calcd for C₁₀H₁₆N₃SO₂:

242.0958. Found: 242.0967.

1

80%. H NMR (300 MHz, CDCl₃) d 9.38, (dd, J = 0.8,

2.2 Hz, 1 H), 8.64 (dd, J = 1.7, 4.8 Hz, 1H), 8.41, (dt,

J = 1.8, 8.0 Hz, 1H), 7.63–7.68 (m, 5H), 7.32–7.42 (m,

6H), 4.54 (t, J = 6.5 Hz, 2H), 3.74 (t, J = 6.1 Hz, 2H),

1.95–2.05 (m, 2H), 1.72–1.81 (m, 2H), 1.05 (s, 9H).

HRMS (ESI+) m/z: Calcd for C₂₇H₃₂N₃O₂SSi:

490.1979. Found: 490.2024.

5.1.6.4. 3-(3-((2-(tert-Butyldiphenylsilyloxy)ethyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(9a). The title compound was synthesized from 8a

according to method A. Yield: 42%. ¹H NMR

(300 MHz, CDCl₃) d 7.60–7.74 (m, 4H), 7.28–7.42 (m,

6H), 7.13–7.19 (m, 1H), 4.56–4.60 (m, 2H), 3.99–4.04

(m, 2H) 3.48–3.54 (m, 2H), 2.58–2.64 (m, 2H), 2.45–

2.50 (m, 5H), 1.06 (s, 9H). HRMS (ESI+) m/z: Calcd

for C₂₆H₃₄N₃O₂SSi: 480.2136. Found: 480.2158.

5.1.7.3. 3-(3-((3-Hydroxypropyl)oxy)-1,2,5-thiadiazol-

4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (10b). The title

compound was synthesized from 9b according to meth-

od C, with a similar work-up as that described for 10a.

Yield: 79%. ¹H NMR (300 MHz, CDCl₃) d 7.01 (m,

1H), 4.59 (t, J = 6.1 Hz, 2H), 3.78 (t, J = 6.1 Hz, 2H),

3.44 (dd, J = 2.4, 4.2 Hz, 2H), 2.57 (t, J = 5.7 Hz, 2H),

2.40–2.48 (m, 5H), 2.33 (br, 1H), 2.07 (p, J = 6.1 Hz,

2H). HRMS (ESI+) m/z: Calcd for C₁₁H₁₈N₃SO₂:

256.1114. Found: 256.1109.

5.1.6.5. 3-(3-((3-(tert-Butyldiphenylsilyloxy)propyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(9b). The title compound was synthesized from 8b

according to method A. Yield: 30%. ¹H NMR

(300 MHz, CDCl₃) d 7.62–7.73 (m, 4H), 7.30–7.43 (m,

6H), 7.03–7.09 (m, 1H), 4.59 (t, J = 6.0 Hz, 2H), 3.70–

3.77 (m, 2H), 3.44–3.50 (m, 2H), 2.58 (t, J = 5.1 Hz,

2H), 2.48–2.54 (m, 5H), 1.93–2.04 (m, 2H), 1.06 (s,

9H). HRMS (ESI+) m/z: Calcd for C₂₇H₃₆N₃O₂SSi:

494.2292. Found: 494.2318.

5.1.8. General procedure for the synthesis of N-tert-

butoxycarbonyl-protected amino acids: method D

5.1.8.1. N-tert-Butoxycarbonyl glycine.¹⁸Glycine

(1.10 g, 14.8 mmol) and NaOH (590 mg, 14.8 mmol)

were stirred in dioxane/H₂O (ꢀ10 mL each) until com-

plete dissolution (ꢀ10 min). This mixture was cooled

to 0 ꢁC at which time Boc₂O (3.53 g, 16.2 mmol) was

added in three equal portions. Stirring continued for

30 min at 0 ꢁC and then for an additional 12 h at room

temperature. The reaction mixture was concentrated

and the resulting residue was redissolved in H₂O

(50 mL). The aqueous layer was washed with diethyl

ether (3· 75 mL), and then acidiﬁed with citric acid to

pH = 4. The aqueous layer was extracted with EtOAc

(3· 30 mL). The combined EtOAc layers were washed

with brine, dried over MgSO₄, and concentrated to yield

N-tert-butoxycarbonyl glycine (2.13 g) as a white solid.

5.1.6.6. 3-(3-((4-(tert-Butyldiphenylsilyloxy)butyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(9c). The title compound was synthesized from 8c

according to method A. Yield: 63%. ¹H NMR

(300 MHz, CDCl₃) d 7.64–7.69 (m, 4H), 7.33–7.43 (m,

6H), 7.02–7.07 (m, 1H), 4.45 (t, J = 6.5 Hz, 2H) 3.67–

3.75 (m, 2H), 3.42–3.46 (m, 2H), 2.56 (t, J = 5.3 Hz,

2H), 2.40–2.46 (m, 5H), 1.89–1.99 (m, 2H), 1.67–1.77

(m, 2H), 1.06 (s, 9H). HRMS (ESI+) m/z: Calcd for

C₂₈H₃₈N₃O₂SSi: 508.2449. Found: 508.2477.

1

Yield: 82%. The H NMR matched previously reported

values.¹⁸

5.1.7. General procedure for TBDPS deprotection (10a–

10c): method C

5.1.8.2.

2-(tert-Butoxycarbonylamino)-1-ethanol

(12a).^37,38N-tert-Butoxycarbonyl glycine (760 mg,

4.33 mmol) was dissolved in THF (20 mL) and cooled

to 0 ꢁC. Sequential addition of Et₃N (0.61 mL,

4.3 mmol) and isobutyl chloroformate (0.56 mL,

4.3 mmol) turned the reaction mixture from clear, to

bright yellow, to rose. The reaction mixture was stirred

for an additional 20 min (0 ꢁC) and then NaBH₄

(491 mg, 13 mmol) was added. Over the next 10 min,

MeOH (30 mL) was added dropwise. The reaction mix-

ture was warmed to room temperature and stirred for an

additional 30 min. The reaction mixture was concen-

trated and extracted with EtOAc (50 mL). The organic

layer was washed with brine, dried over MgSO₄and

concentrated. Column chromatography eluting with a

gradient of CH₂Cl₂/MeOH (9:1–6:1) aﬀorded title com-

5.1.7.1. 3-(3-((4-Hydroxybutyl)oxy)-1,2,5-thiadiazol-

4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (10c). To a stir-

ring solution of 9c (690 mg, 1.36 mmol) in THF (5 mL)

at 0 ꢁC was added TBAF in THF (1.77 mL of a 1.0 M

solution, 1.77 mmol). The reaction mixture was stirred

at room temperature for 3 h and then concentrated.

The resulting residue was puriﬁed by silica gel column

chromatography (9:1:0.2 CH₂Cl₂/MeOH/Et₃N) to fur-

nish 10c as a yellow solid (169 mg). Yield: 46%. ¹H

(300 MHz, CDCl₃) d 7.05 (m, 1H), 4.47 (t, J = 6.5 Hz,

2H), 3.70 (t, J = 6.4 Hz, 2H), 3.45 (dd, J = 2.5, 4.4 Hz,

2H), 2.58 (t, J = 5.8 Hz, 2H), 2.44–2.47 (m, 5H), 1.88–

1.98 (m, 2H), 1.66–1.77 (m, 2H). HRMS (ESI+) m/z:

Calcd for C₁₂H₂₀N₃SO₂: 270.1271. Found: 270.1223.

1

pound 12a. Yield: 45%. H NMR (300 MHz, CDCl₃) d

5.1.7.2. 3-(3-((2-Hydroxyethyl)oxy)-1,2,5-thiadiazol-

4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (10a). The title

compound was synthesized from 9a according to

4.94 (br, 1H), 3.72 (t, J = 4.5 Hz, 2H), 3.24–3.30 (m,

2H), 2.71 (br, 1H), 1.45 (s, 9H). HRMS (APCI+) m/z:

Calcd for C₇H₁₆NO₃: 162.1125. Found: 162.1164.

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1387

5.1.8.3. 4-(tert-Butoxycarbonylamino)-1-butanol (12b).

Starting from 4-aminobutanoic acid (GABA), N-tert-

butoxycarbonyl-GABA was synthesized according to

method D (88% yield).¹⁸The acid was reduced using a

similar mixed anhydride reduction as 12a, except that

ethyl chloroformate was utilized instead of isobutyl

chloroformate. Additionally, the NaBH₄(3 equiv) was

added as a solution in MeOH and the resulting mixture

was allowed to stir at room temperature for 2 h.²⁰The

reaction mixture was concentrated and partitioned be-

tween EtOAc and H₂O. The organic layer was sepa-

rated, washed with brine, dried over MgSO₄, and

concentrated to aﬀord 12b. Yield: 65%. ¹H NMR

(300 MHz, CDCl₃) d 4.72 (br, 1H), 3.60–3.69 (m, 2H),

3.08–3.19 (m, 2H), 2.12–2.20 (m, 1H), 1.51–1.61 (m,

4H), 1.44 (s, 9H). HRMS (APCI+) m/z: Calcd for

C₉H₂₀NO₃: 190.1438. Found: 190.1452.

NMR (300 MHz, CDCl₃) d 3.28 (t, J = 6.0 Hz, 2H),

2.84 (s, 3H), 2.35 (t, J = 7.0 Hz, 2H), 1.80–1.87 (m,

2H), 1.45 (s, 9H). HRMS (ESIÀ) m/z: Calcd for

C₁₀H₁₈NO₄: 216.1241. Found: 216.1252.

5.1.8.8. 4-(tert-Butoxycarbonyl(methyl)amino)-1-buta-

nol (17).²⁴To a solution of 16 (502 _Åmg, 2.47 mmol) in

THF (10 mL) at 0 ꢁC was added BH₃THF (2.6 mL of a

1.0 M solution). The reaction was allowed to warm to

room temperature and was stirred for an additional

18 h. The reaction mixture was then chilled to 0 ꢁC and

ꢀ3 mL of H₂O was added. The reaction mixture was con-

centrated, extracted with EtOAc, and washed with a citric

acid solution (pH = 3.5). The organic layer was dried over

MgSO₄and concentrated. Column chromatography elut-

ing with a gradient of CH₂Cl₂/MeOH (9:1–4:1) aﬀorded

compound 17 as a colorless oil (390 mg). Yield: 83%. ¹H

NMR (300 MHz, CDCl₃) d 3.66 (t, J = 6.0 Hz, 2H),

3.23 (t, J = 6.6 Hz, 2H), 2.83 (s, 3H), 1.78 (br, 1H),

1.51–1.60 (m, 4H), 1.44 (s, 9H). HRMS (APCI+) m/z:

Calcd for C₁₀H₂₂NO₃: 204.1594. Found: 204.1582.

5.1.8.4. 6-(tert-Butoxycarbonylamino)-1-hexanol (12c).

Starting from 6-aminohexanoic acid, the amino group

was Boc-protected utilizing method D (89% yield).¹⁸

The acid was reduced using the procedure detailed for

1

12b.²⁰Yield: 41%. H NMR (300 MHz, CDCl₃) d 4.52

5.1.9. General procedure for preparation of compounds

18a–e, 21, 24, and 25: method E

(br, 1H), 3.64 (t, J = 6.5 Hz, 2H), 3.06–3.16 (m, 2H),

1.28–1.60 (m, 17H). HRMS (APCI+) m/z: Calcd for

C₁₁H₂₄NO₃: 218.1756. Found: 218.1747.

5.1.9.1. 3-(3-((4-(tert-Butoxycarbonyl)aminobutyl)oxy)-

1,2,5-thiadiazol-4-yl) pyridine (18c). To a stirring solution

of 12b (250 mg, 1.32 mmol) in THF (ꢀ15 mL) at 0 ꢁC

was added NaH (105 mg, 2.63 mmol). The reaction

mixture was reﬂuxed for 1 h and then cooled to 0 ꢁC.

Intermediate 3 (260 mg, 1.32 mmol) in THF (10 mL)

was added. The reaction mixture was reﬂuxed for 18 h,

cooled, and then concentrated. The residue was taken

up in CH₂Cl₂(50 mL/mmol), washed with 5% NaOH,

brine, dried over Na₂SO₄, and concentrated. The result-

ing residue was puriﬁed using silica gel column chroma-

tography eluting with CH₂Cl₂/MeOH (9:1). Compound

18c was isolated as a brown oil (205 mg). Yield: 44%. ¹H

NMR (300 MHz, CDCl₃) d 9.38 (d, J = 0.8 Hz, 1H),

8.64 (dd, J = 1.5, 4.8 Hz, 1H), 8.43–8.50 (m, 1H), 7.45

(dd, J = 5.0, 7.6 Hz, 1H), 4.70 (br, 1H), 4.53 (t,

J = 6.4 Hz, 2H), 3.15–3.23 (m, 2H), 1.86–1.96 (m, 2H),

1.62–1.72 (m, 2H), 1.41 (s, 9H). HRMS (ESI+) m/z:

Calcd for C₁₆H₂₃N₄O₃S: 351.1485. Found: 351.1531.

5.1.8.5.

3-(tert-Butoxycarbonylamino)-1-propanol

(14a).²¹To a solution of 3-aminopropan-1-ol (628 mg,

8.50 mmol) and Et₃N (1.17 mL, 8.50 mmol) in CH₂Cl₂

(10 mL) at 0 ꢁC, Boc₂O (1.69 g, 7.72 mmol) was added.

The reaction mixture was stirred at 0 ꢁC for 30 min

and then stirred for an additional 12 h at room temper-

ature. The reaction mixture was concentrated and the

residue was dissolved in Et₂O (50 mL). The organic

phase was washed with citric acid (pH ꢀ 4.0), saturated

NaHCO₃, and brine. The ethereal layer was dried over

MgSO₄and concentrated to yield 14a (1.22 g). Yield:

90%. ¹H NMR (300 MHz, CDCl₃) d 4.98 (br, 1H),

3.66 (q, J = 6.0 Hz, 2H), 3.39 (t, J = 6.0 Hz, 1H), 3.28

(q, J = 6.0 Hz, 2H), 1.67 (p, J = 6.0 Hz, 2H), 1.45 (s,

9H). HRMS (APCI+) m/z: Calcd for C₈H₁₈NO₃:

176.1281. Found: 176.1043.

5.1.8.6.

5-(tert-Butoxycarbonylamino)-1-pentanol

5.1.9.2. 3-(3-((2-(tert-Butoxycarbonyl)aminoethyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (18a). The title compound

was synthesized according to method E. Instead of

reﬂuxing for 18 h, however, the reaction mixture was

stirred at room temperature (18 h). Column chromatog-

raphy eluting with CH₂Cl₂/MeOH (9:1) aﬀorded com-

(14b).³⁹The title compound was synthesized in an anal-

ogous manner to aﬀord propanol derivative 14a. Yield:

1

99%. H NMR (300 MHz, CDCl₃) d 4.60 (br, 1H), 3.65

(q, J = 5.8 Hz, 2H), 3.11–3.16 (m, 2H), 1.69 (t,

J = 5.1 Hz, 1H), 1.50–1.59 (m, 6H), 1.44 (s, 9H). HRMS

(APCI+) m/z: Calcd for C₁₀H₂₂NO₃: 204.1594. Found:

204.1590.

1

pound 18a. Yield: 30%. H NMR (300 MHz, CDCl₃)

d 9.37 (d, J = 1.6 Hz, 1H), 8.67 (dd, J = 1.7, 4.8 Hz,

1H), 8.44 (dt, J = 1.6, 8.1 Hz, 1H), 7.42 (ddd, J = 0.9,

4.8, 8.0 Hz, 1H), 5.04 (br, 1H), 4.62, (t, J = 5.3 Hz,

2H), 3.61–3.69 (m, 2H), 1.43 (s, 9H). HRMS (ESI+)

m/z: Calcd for C₁₄H₁₉N₄O₃S: 323.1172. Found:

323.1177.

5.1.8.7. 4-(tert-Butoxycarbonyl(methyl)amino)butyric

acid (16).²³A solution of concentrated HCl (20 mL)

and 1-methyl-2-pyrrolidinone (10 mL) was reﬂuxed for

18 h. The reaction mixture was cooled and concentrated.

The resulting white solid matched previously reported

NMR data for 4-(methylamino)butanoic acid hydro-

chloride salt and was used without further puriﬁca-

tion.⁴⁰The Boc group was installed using method D

with an additional equivalent of NaOH to neutralize

5.1.9.3. 3-(3-((3-(tert-Butoxycarbonyl)aminopropyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (18b). The title compound

was synthesized according to method E with the same

modiﬁcation as that which was denoted in 18a. Yield:

1

the HCl salt of the precursor. Yield from 15: 52%. H

50%. H NMR (300 MHz, CDCl₃) d 9.38 (dd, J = 0.8,

1388

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

2.4 Hz, 1H), 8.66 (dd, J = 1.7, 4.8 Hz, 1H), 8.44 (dt,

J = 1.8, 7.2 Hz, 1H), 7.42 (ddd, J = 0.9, 4.8, 8.0 Hz,

1H), 4.64 (t, J = 6.0 Hz, 2H), 5.73 (br, 1H), 3.38–3.45

(m, 2H), 2.12–2.22 (m, 2H), 1.45 (s, 9H). HRMS

(ESI+) m/z: Calcd for C₁₅H₂₁N₄O₃S: 337.1329. Found:

337.1242.

5.1.9.9. 3-(3-((5-(tert-Butoxycarbonyl)aminopentyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(19d). The title compound was synthesized from 18d

according to method A. Yield: 33%. ¹H NMR

(300 MHz, CDCl₃) d 7.00–7.07 (m, 1H), 4.77 (br, 1H),

4.53 (t, J = 6.0 Hz, 2H), 3.40–3.46 (m, 2H), 3.11–3.20

(m, 2H), 2.57 (t, J = 5.7 Hz, 2H), 2.42–2.47 (m, 5H),

1.82–1.94 (m, 2H), 1.47–1.65 (m, 4H), 1.44 (s, 9H).

HRMS (ESI+) m/z: Calcd for C₁₈H₃₁N₄O₃S: 383.2111.

Found: 383.2159.

5.1.9.4. 3-(3-((5-(tert-Butoxycarbonyl)aminopentyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (18d). The title compound

was synthesized according to method E. Yield: 72%.

¹H NMR (300 MHz, CDCl₃) d 9.37–9.41 (m, 1H), 8.65

(dd, J = 1.7, 4.8 Hz, 1H), 8.39–8.44 (m, 1H), 7.37–7.43

(m, 1H), 4.90 (br, 1H), 4.50–4.54 (m, 2H), 3.12–3.26

(m, 2H), 1.86–1.97 (m, 2H), 1.50–1.64 (m, 4H), 1.43 (s,

9H). HRMS (ESI+) m/z: Calcd for C₁₇H₂₅N₄O₃S:

365.1642. Found: 365.1673.

5.1.9.10. 3-(3-((6-(tert-Butoxycarbonyl)aminohexyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(19e). The title compound was synthesized from 18a

according to method A. Column chromatography elut-

ing with CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 19e as

1

a brown oil. Yield: 37%. H NMR (300 MHz, CDCl₃)

5.1.9.5. 3-(3-((6-(tert-Butoxycarbonyl)aminohexyl)oxy)-

1,2,5-thiadiazol-4-yl)pyridine (18e). The title compound

was synthesized according to method E. Yield: 85%.

¹H NMR (300 MHz, CDCl₃) d 9.38 (d, J = 0.8 Hz, 1

H), 8.64 (dd, J = 1.2, 4.7 Hz, 1H), 8.44 (ddd, J = 1.9,

2.1, 8.1 Hz), 7.43 (ddd, J = 0.8, 4.9, 8.0 Hz), 4.62, (br,

1H), 4.50 (t, J = 6.6 Hz, 2H), 3.06–3.16 (m, 2H), 1.83–

1.93 (m, 2H), 1.45–1.55 (m, 4H), 1.42 (s, 9H). HRMS

(ESI+) m/z: Calcd for C₁₈H₂₇N₄O₃S: 379.1798. Found:

379.1813.

d 7.00–7.05 (m, 1H), 4.65 (br, 1H), 4.40 (t, J = 6.6 Hz,

2H), 3.43–3.47 (m, 2H), 3.06–3.10 (m, 2H), 2.58 (t,

J = 5.9 Hz, 2H), 2.39–2.45 (m, 5H), 1.75–1.86 (m, 2H),

1.36–1.50 (m, 15H). HRMS (ESI+) m/z: Calcd for

C₁₉H₃₃N₄O₃S: 397.2268. Found: 397.2293.

5.1.10. Boc-deprotection reactions. The Boc group was

removed via one of two diﬀerent methods. One method

involved stirring the starting material in a 50/50 mixture

of TFA and CH₂Cl₂.⁴¹After stirring for 4 h the reaction

mixture was concentrated. Alternatively, the Boc group

was removed by stirring in a solution of 4.0 M HCl in

dioxane. Similarly, the reaction mixture was stirred for

4 h and was then concentrated.

5.1.9.6. 3-(3-((2-(tert-Butoxycarbonyl)aminoethyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(19a). The title compound was synthesized from 18a

according to method A. Column chromatography elut-

ing with CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 19a as

5.1.11. 3-(3-((2-Aminoethyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (20a) 2^ÅTFA salt.

¹H NMR (300 MHz, CDCl₃) d 7.00–7.05 (m, 1H),

4.30–4.40 (m, 2H), 3.81–3.92 (m, 2H), 3.33–3.52 (m,

2H), 3.03–3.17 (m, 2H), 2.90–2.97 (m, 2H), 2.84–2.89

(m, 5H). HRMS (ESI+) m/z: Calcd for C₁₀H₁₇N₄OS:

241.1118. Found: 241.1112.

1

a brown oil. Yield: 46%. H NMR (300 MHz, CDCl₃)

d 7.00–7.05 (m, 1H), 4.89 (br, 1H), 4.52 (t, J = 5.4 Hz,

2H), 3.57–3.63 (m, 2H), 3.47–3.50 (m, 2H), 2.62 (t,

J = 5.7 Hz), 2.45–2.50 (m, 5H), 1.42 (s, 9H). HRMS

(ESI+) m/z: Calcd for C₁₅H₂₅N₄O₃S 341.1642. Found:

341.1686.

5.1.9.7. 3-(3-((3-(tert-Butoxycarbonyl)aminopropyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(19b). The title compound was synthesized from 18b

according to method A. Column chromatography elut-

ing with CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 19b as

5.1.12. 3-(3-((3-Aminopropyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (20b). This was orig-

inally made as the TFA salt, but since it did not easily

recrystallize, it was dissolved in 1 M K₂CO₃, extracted,

concentrated, and stored as its freebase. ¹H NMR

(300 MHz, CDCl₃) d 7.00–7.06 (m, 1H), 4.54 (t,

J = 5.1 Hz, 2H), 3.62–3.76 (m, 2H), 3.41–3.46 (m, 2H),

2.86–2.94, (m, 2H), 2.53–2.60 (m, 2H) 2.40–2.48 (m,

5H), 1.94–2.04 (m, 2H). HRMS (ESI+) m/z: Calcd for

C₁₁H₁₉N₄OS: 255.1274. Found: 255.1234.

1

a brown oil. Yield: 16%. H NMR (300 MHz, CDCl₃)

d 7.00–7.05 (m, 1H), 4.76 (br, 1H), 4.52 (t, J = 6.2 Hz,

2H), 3.44–3.48 (m, 2H), 3.26–3.34 (m, 2H), 2.59 (t,

J = 5.3 Hz, 2H), 2.44–2.48 (m, 5H), 2.00–2.10 (m, 2H),

1.44 (s, 9H). HRMS (ESI+) m/z: Calcd for

C₁₆H₂₇N₄O₃S: 355.1798. Found: 355.1814.

5.1.13. 3-(3-((4-Aminobutyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (20c) 2^ÅHCl salt.

Boc-deprotection was done using 4.0 M dioxane in

5.1.9.8. 3-(3-((4-(tert-Butoxycarbonyl)aminobutyl)oxy)-

1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine

(19c). The title compound was synthesized from 18c

according to method A. Column chromatography elut-

ing with CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 19c as

1

HCl solution. H NMR (300 MHz, D₂O) d 6.88–6.94

(m, 1H), 4.24–4.29 (m, 2H), 3.68–3.78 (m, 2H), 3.27–

3.37 (m, 2H), 3.00–3.05 (m, 2H), 2.70–2.77 (m, 7H),

1.49–1.68 (m, 4H). HRMS (ESI+) m/z: Calcd for

C₁₂H₂₁N₄OS: 269.1431. Found: 269.1444.

1

a brown oil. Yield: 16%. H NMR (300 MHz, CDCl₃)

d 7.03–7.08 (m, 1H), 4.62 (br, 1H), 4.47 (t, J = 6.5 Hz,

2H), 3.51–3.55 (m, 2H), 3.15–3.24 (m, 2H), 2.67 (t,

J = 5.6 Hz, 2H), 2.49–2.54 (m, 5H), 1.83–1.94 (m, 2H),

1.60–1.71 (m, 2H), 1.45 (s, 9H). HRMS (ESI+) m/z:

Calcd for C₁₇H₂₉N₄O₃S: 369.1955. Found: 369.1956.

5.1.14. 3-(3-((5-Aminopentyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (20d) 2^ÅHCl salt.

Boc-deprotection was done using 4.0 M dioxane in

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1389

1

HCl solution. H NMR (300 MHz, D₂O) d 7.06–7.11

J = 1.8, 4.8 Hz, 1H), 8.40 (ddd, J = 1.8, 2.4, 8.1 Hz,

1H), 7.38 (ddd, J = 0.9, 5.1, 8.1 Hz, 1H), 7.13–7.30 (m,

5 H), 4.53 (t, J = 6.3 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H),

1.80–1.99 (m, 4H). HRMS (ESI+) m/z: Calcd for

C₁₇H₁₈N₃OS: 312.1165. Found: 312.1187.

(m, 1H), 4.42 (t, J = 4.5 Hz, 2H), 3.68–3.75 (m, 2H),

3.57–3.65 (m, 2H), 2.80–2.90 (m, 2H), 2.59–2.67 (m,

5H), 2.48–2.56 (m, 2H), 1.77–1.88 (m, 2H), 1.42–1.64

(m, 4H) HRMS (ESI+) m/z: Calcd for C₁₃H₂₃N₄OS:

283.1587. Found: 283.1604.

5.1.21. 3-(3-((3-Phenyl-2-propyn)oxy)-1,2,5-thiadiazol-4-

yl)-1,2,5,6-tetrahydro-1-methylpyridine (26).¹³The title

compound was synthesized from 24 according to meth-

od A. Column chromatography eluting with CH₂Cl₂/

MeOH/Et₃N (9:1:0.2) aﬀorded 26. Yield: 11%. ¹H

NMR (300 MHz, CDCl₃) d 7.45–7.50 (m, 2H), 7.31–

7.37 (m, 3H), 7.10–7.16 (m, 1H), 5.30 (s, 2H), 3.44–

3.47 (m, 2H), 2.59 (t, J = 5.1 Hz, 2H), 2.47–2.51 (m,

5H). HRMS (ESI+) m/z: Calcd for C₁₇H₁₈N₃OS:

312.1165. Found: 312.1252.

5.1.15. 3-(3-((6-Aminohexyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (20e). Boc-deprotec-

tion was done using 4.0 M dioxane in HCl solution.

¹H NMR (300 MHz, D₂O) d 7.03–7.08 (m,1H), 4.28–

4.34 (m, 2H), 3.82–3.91 (m, 2H), 3.05–3.14 (m, 2H),

2.85–2.90, (m, 5H), 2.81 (t, J = 5.0 Hz, 2H), 2.49–2.59

(m, 2H), 1.65–1.75 (m, 2H), 1.24–1.55 (m, 6H). HRMS

(ESI+) m/z: Calcd for C₁₄H₂₅N₄OS: 297.1744. Found:

297.1779.

5.1.16. 3-(3-((4-(tert-Butoxycarbonyl(methyl)amino)butyl)-

oxy)-1,2,5-thiadiazol-4-yl)pyridine (21). The title com-

pound was synthesized according to method E. Yield:

5.1.22. 3-(3-((4-Phenylbutyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (27) HCl salt.¹³

The title compound was synthesized from 24 according

to method A. Column chromatography eluting with

CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded a mixture of 27

and 4-phenylbutan-1-ol. This mixture was dissolved in

HCl/EtOH, stirred for 30 min, and concentrated. The

resulting oil was repeatedly washed with Et₂O, which

eventually led to the precipitation of the HCl salt of

1

82%. H NMR (300 MHz, CDCl₃) d 9.37 (dd, J = 0.8,

2.3 Hz, 1H), 8.64 (dd, J = 1.7, 4.8 Hz, 1H), 8.40 (dt,

J = 1.9, 7.2 Hz, 1H), 7.38 (ddd, J = 0.8, 4.8, 7.2 Hz,

1H), 4.55 (t, J = 6.4 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H),

2.84 (s, 3H), 1.83–1.92 (m, 2H), 1.66–1.75 (m, 2H),

1.43 (s, 9H). HRMS (ESI+) m/z: Calcd for

C₁₇H₂₅N₄O₃S: 365.1642. Found: 365.1826.

1

27. Yield: 19%. Freebase NMR: H NMR (300 MHz,

CDCl₃) d 7.26–7.32 (m, 2H), 7.16–7.23 (m, 3H), 7.02–

7.07 (m, 1H), 4.46 (t, J = 6.3 Hz, 2H), 3.44–3.48 (m,

2H), 2.70 (t, J = 7.8 Hz, 2H), 2.57 (t, J = 5.1 Hz. 2H),

2.45–2.48 (m, 5H), 1.76–1.94 (m, 4H). HRMS (ESI+)

m/z: Calcd for C₁₈H₂₄N₃OS: 330.1625. Found:

330.1681.

5.1.17. 3-(3-((4-(tert-Butoxycarbonyl(methyl)amino)butyl)-

oxy)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyri-

dine (22). The title compound was synthesized from 21

according to method A. Yield: 30%. ¹H NMR

(300 MHz, CDCl₃) d 7.02–7.06 (m, 1H), 4.46 (t,

J = 6.3 Hz, 2H), 3.42–3.46 (m, 2H), 3.22–3.33 (m, 2H),

2.84 (s, 3H), 2.58 (t, J = 5.4 Hz, 2H), 2.41–2.47 (m,

5H), 1.79–1.89 (m, 2H), 1.63–1.73 (m, 2H), 1.45 (s,

9H). HRMS (ESI+) m/z: Calcd for C₁₈H₃₁N₄O₃S:

383.2111. Found: 383.2042.

5.1.23. 3-(3-((4-Chlorobutyl)oxy)-1,2,5-thiadiazol-4-yl)-

1,2,5,6-tetrahydro-1-methylpyridine (28). To a stirring

solution of 10c (29 mg, 0.11 mmol) in CH₂Cl₂(5 mL)

at 0 ꢁC was added SOCl₂(0.20 mL, 2.8 mmol). The reac-

tion was warmed to room temperature and stirred for

12 h. After cooling to 0 ꢁC, H₂O (ꢀ5 mL) was added,

followed by K₂CO₃, until the pH of the aqueous layer

reached ꢀ12. To this mixture was added CH₂Cl₂

(ꢀ10 mL). The separated aqueous layer was further ex-

tracted with CH₂Cl₂(3· 10 mL). The combined organics

were washed with brine, dried over Na₂SO₄, and con-

centrated to yield the title compound (32 mg). Yield:

100%. ¹H NMR (300 MHz, CDCl₃) d 7.04 (m, 1H),

4.49 (t, J = 6.1 Hz, 2H), 3.63 (t, J = 6.2 Hz, 2H), 3.49

(dd, J = 2.4, 4.3 Hz, 2H), 2.62 (t, J = 5.8 Hz, 2H),

2.47–2.53 (m, 5 H), 1.91–2.08 (m, 4H). HRMS (ESI+)

m/z: Calcd for C₁₂H₁₉N₃SOCl: 288.0932. Found:

288.0897.

5.1.18. 3-(3-((4-Methylaminobutyl)oxy)-1,2,5-thiadiazol-

4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (23). Com-

pound 22 was Boc-deprotected with TFA in CH₂Cl₂.

1

Yield: 34%. H NMR (300 MHz, CDCl₃) d 7.00–7.06

(m, 1H), 4.46 (t, J = 6.5 Hz, 2H), 3.41–3.47 (m, 5H),

2.79 (t, J = 4.5 Hz, 2H), 2.58 (t, J = 5.4 Hz, 2H), 2.42–

2.46 (m, 5H), 1.87–1.94 (m, 2H), 1.70–1.78 (m, 2H).

HRMS (ESI+) m/z: Calcd for C₁₃H₂₃N₄OS: 283.1587.

Found: 283.1575.

5.1.19. 3-(3-((3-Phenyl-2-propyn)oxy)-1,2,5-thiadiazol-4-

yl)pyridine (24). The title compound was synthesized

according to method E. Yield: 75%. ¹H NMR

(300 MHz, CDCl₃) d 9.43 (dd, J = 0.9, 2.4 Hz, 1H),

8.67 (dd, J = 1.8, 4.8 Hz, 1H), 8.46 (ddd, J = 1.8, 2.4,

7.8 Hz, 1H), 7.45–7.51 (m, 2H), 7.41 (ddd, J = 0.9, 5.1,

8.1 Hz, 1H), 7.30–7.37 (m, 3H), 5.39 (s, 2H). HRMS

(ESI+) m/z: Calcd for C₁₆H₁₂N₃OS: 294.0696. Found:

294.0721.

5.1.24. 3-(3-(4-(1H-imidazol-1-yl)butoxy)-1,2,5-thiadiazol-

4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (29). To a stir-

ring solution of imidazole (10.4 mg, 0.153 mmol)

in DMF (5 mL) at 0 ꢁC was added NaH (6.1 mg,

0.15 mmol). The reaction mixture was reﬂuxed for 1 h

and then chilled to 0 ꢁC. Compound 28 (22 mg,

0.076 mmol) in DMF (ꢀ3 mL) was added and the reac-

tion was reﬂuxed for 18 h. The reaction mixture was

cooled and concentrated. The resulting residue was dis-

solved in CH₂Cl₂(30 mL) and subsequently washed

5.1.20. 3-(3-((4-Phenylbutyl)oxy)-1,2,5-thiadiazol-4-yl)pyr-

idine (25). The title compound was synthesized accord-

ing to method E. Yield: 82%. ¹H NMR (300 MHz,

CDCl₃) d 9.39 (dd, J = 0.9, 2.4 Hz, 1H), 8.64 (dd,

1390

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

with 5% NaOH and brine. The organic layer was dried

over Na₂SO₄and concentrated to yield crude product.

Column chromatography on silica gel eluting with

CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 29 (3.2 mg).

Yield: 13%. ¹H NMR (600 MHz, CDCl₃) d 7.48 (s,

1H), 7.06 (s, 1H), 6.96–7.01 (m, 1H), 6.91 (s, 1H), 4.45

(t, J = 6.2 Hz, 2H), 4.02 (t, J = 6.8 Hz, 2H), 3.43–3.45

(m, 2H), 2.57 (t, J = 6.0 Hz, 2H), 2.45–2.49 (m, 5H),

1.91–1.99 (m, 2H), 1.80–1.88 (m, 2H). HRMS (ESI+)

m/z: Calcd for C₁₅H₂₂N₅SO: 320.1540. Found:

320.1496.

5H), 2.38 (t, J = 7.1 Hz, 2H), 1.75–1.92 (m, 4H), 1.25

(t, J = 7.2 Hz, 3H). HRMS (ESI+) m/z: Calcd for

C₁₅H₂₄N₃SO₃: 326.1533. Found: 326.1572.

5.1.27. 3-(3-Chloro-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahy-

dro-1-methylpyridine (32). The title compound was syn-

thesized from 3 according to method A. Column

chromatography eluting with CH₂Cl₂/MeOH/Et₃N

(9:1:0.2) aﬀorded 32 as a brown oil. Yield: 31%. ¹H

NMR (300 MHz, CDCl₃) d 6.91–6.96 (m, 1H), 3.33–

3.37 (m, 2H), 2.53 (t, J = 5.7 Hz, 2H), 2.38–2.45 (m,

5H). HRMS (ESI+) m/z: Calcd for C₈H₁₁N₃SCl:

216.0357. Found: 216.0356.

5.1.25. 3-(3-(4-(1H-pyrrol-yl)butoxy)-1,2,5-thiadiazol-4-

yl)-1,2,5,6-tetrahydro-1-methylpyridine (30). To a stir-

ring solution of pyrrole (15.0 mg, 0.224 mmol) in

DMF (5 mL) at 0 ꢁC was added NaH (9.0 mg,

0.23 mmol). The reaction mixture was reﬂuxed for 1 h

and then chilled to 0 ꢁC. Compound 28 (20.1 mg,

0.070 mmol) in DMF (ꢀ3 mL) was added and the reac-

tion was reﬂuxed for 18 h. The reaction mixture was

cooled and concentrated. The resulting residue was dis-

solved in CH₂Cl₂(30 mL) and subsequently washed

with 5% NaOH and brine. The organic layer was dried

over Na₂SO₄and concentrated to yield crude product.

Column chromatography on silica gel eluting with

CH₂Cl₂/MeOH/Et₃N (9:1:0.2) aﬀorded 30 (4.3 mg).

5.1.28. Ethyl 2-(4-(1-methyl-1,2,5,6-tetrahydropyridin-3-

yl)-1,2,5-thiadiazol-3-yloxy)acetate (33). The title com-

pound was synthesized according to method E, with

modiﬁcation to the amounts of glycolic acid ethyl ester

(10 equiv) and NaH (10 equiv) used. Column chroma-

tography on alumina (basic, Brockman Activity I) elut-

ing with hexane/EtOAc (3:1) aﬀorded the title

1

compound. Yield: 20%. H NMR (300 MHz, CDCl₃)

d 7.11–7.17 (m, 1H), 5.00 (s, 2H), 4.27 (q, J = 7.2 Hz,

2H), 3.44–3.47 (m, 2H), 2.57 (t, J = 4.8 Hz, 2H), 2.43–

2.47 (m, 5H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI+)

m/z: Calcd for C₁₂H₁₈N₃SO₃: 284.1063. Found:

284.1041.

1

Yield: 19%. H NMR (300 MHz, CDCl₃) d 6.99–7.05

(m, 1H), 6.63–6.68 (m, 2H), 6.12–6.16 (m, 2H), 4.44 (t,

J = 6.0 Hz, 2H), 3.96 (t, J = 7.0 Hz, 2H), 3.41–3.46 (m,

2H), 2.57 (t, J = 5.5 Hz, 2H), 2.41–2.47 (m, 5H), 1.88–

2.06 (m, 4H). HRMS (ESI+) m/z: Calcd for

C₁₆H₂₃N₄SO: 319.1587. Found: 319.1539.

5.1.29.

2-(4-(1-Methyl-1,2,5,6-tetrahydropyridin-3-yl)-

1,2,5-thiadiazol-3-yloxy)acetic acid (34) lithium salt. To

a stirring solution of 33 (23.0 mg, 0.082 mmol) in

CH₃CN/H₂O (5 mL each) was added LiOH^ÅH₂O

(3.40 mg, 0.082 mmol). The reaction mixture was heated

at 50 ꢁC for 2 h and then concentrated to aﬀord a yellow

5.1.26. Ethyl 5-(4-(1-methyl-1,2,5,6-tetrahydropyridin-3-

yl)-1,2,5-thiadiazol-3-yloxy)pentanoate (31). To a stirring

solution of the sodium salt of 5-hydroxypentanoic acid

(185 mg, 1.32 mmol) in THF (20 mL) at 0 ꢁC was added

NaH (106 mg, 2.64 mmol). The reaction mixture was re-

ﬂuxed for 1 h and then cooled to 0 ꢁC at which time

intermediate 3 (261 mg, 1.32 mmol) in THF (5 mL)

was added. The reaction was reﬂuxed for an additional

18 h and then concentrated in vacuo. The resulting res-

idue was partitioned between Et₂O and H₂O. The sepa-

rated aqueous layer was acidiﬁed to pH = 2, washed

with Et₂O (3· 30 mL), and concentrated. The resulting

residue was suspended in DMF (10 mL) and ﬁltered.

To the ﬁltrate, K₂CO₃(365 mg, 2.64 mmol) and iodo-

methane (0.165 mL, 2.64 mmol) were added. This mix-

ture was stirred at room temperature for 18 h and then

concentrated. The resulting residue was dissolved in

CH₂Cl₂(30 mL), washed with water (30 mL) and brine.

The aqueous layer was further extracted with CH₂Cl₂

(2· 30 mL). The combined organics were concentrated,

dissolved in EtOH (20 mL), and chilled to 0 ꢁC. To this

solution was added NaBH₄(110 mg, 2.9 mmol). The

reaction mixture was reﬂuxed for 4 h and then concen-

trated. The residue was taken up in CH₂Cl₂, washed

with water, brine, dried over Na₂SO₄, and concentrated.

Column chromatography eluting with CH₂Cl₂/MeOH/

Et₃N (15:1:0.2) aﬀorded 31 as a yellow solid (16 mg).

Yield: 4%. ¹H NMR (300 MHz, CDCl₃) d 7.05 (m,

1H), 4.46 (t, J = 6.1 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H),

3.44–3.48 (m, 2H), 2,59 (t, J = 5.4 Hz, 2H), 2.47 (m,

1

solid (21.2 mg). Yield: 99%. H NMR (600 MHz, D₂O)

d 7.00–7.06 (m, 1H), 4.68 (s, 2H), 3.29–3.34 (m, 2H),

2.52–2.58 (m, 2H), 2.28–2.36 (m, 5H). HRMS (ESI+)

m/z: Calcd for C₁₀H₁₄N₃SO₃: 256.0750. Found:

256.0686. HRMS (ESIÀ) m/z: Calcd for C₁₀H₁₂N₃SO₃:

254.0605. Found: 254.0554.

5.2. Biological assays

[³H] N-Methyl scopolamine ([³H]NMS) was purchased

from Perkin-Elmer Life Sciences (Boston, MA).

Atropine (racemic mixture) was purchased from Sig-

ma–Aldrich (St. Louis, MO). Xanomeline analogs

(LY1–LY3) were a kind gift from Eli Lilly & Co. (Indi-

anapolis, IN). The NNC series of ligands were

obtained from the El-Fakahany laboratory.⁴²The com-

ponents of the buﬀered solutions were purchased from

multiple commercial sources. Experiments were per-

formed on Chinese hamster ovary (CHO) cells stably

transfected with the human gene for the muscarinic

M₁receptor (kindly provided by Dr. M. Brann, Acadia

Pharmaceuticals, Inc., San Diego, CA). Cells were

grown in plastic dishes in Dulbecco’s modiﬁed Eagle’s

medium with 10% bovine calf serum and 50 lg/mL

geneticin. They were harvested by mild trypsinization

4 days after subculturing and washed twice by centrifu-

gation (3 min at 300g) and resuspension in a Hepes

medium (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl₂,

1 mM MgSO₄, 25 mM glucose, 20 mM Hepes, 58 mM

B. E. Kane et al. / Bioorg. Med. Chem. 16 (2008) 1376–1392

1391

12. Jakubik, J.; Tucek, S.; El-Fakahany, E. E. J. Pharmacol.

Exp. Ther. 2002, 301, 1033–1041.

sucrose, with pH adjusted to 7.4 and osmolarity to

340 mOsM).

13. Sauerberg, P.; Jeppesen, L.; Olesen, P. H.; Sheardown, M.

J.; Fink-Jensen, A.; Rasmussen, T.; Rimvall, K.; Shannon,

H. E.; Bymaster, F. P.; DeLapp, N. W.; Calligaro, D. O.;

Ward, J. S.; Whitesitt, C. A.; Thomsen, C. Bioorg. Med.

Chem. Lett. 1998, 8, 2897–2902.

14. Sauerberg, P.; Olesen, P. H.; Nielsen, S.; Treppendahl, S.;

Sheardown, M. J.; Honore, T.; Mitch, C. H.; Ward, J. S.;

Pike, A. J.; Bymaster, F. P., et al. J. Med. Chem. 1992, 35,

2274–2283.

15. Rajeswaran, W. G.; Cao, Y.; Huang, X. P.; Wroblewski,

M. E.; Colclough, T.; Lee, S.; Liu, F.; Nagy, P. I.; Ellis, J.;

Levine, B. A.; Nocka, K. H.; Messer, W. S., Jr. J. Med.

Chem. 2001, 44, 4563–4576.

16. Yu, C.; Liu, B.; Hu, L. Tetrahedron Lett. 2000, 41, 4281–

4286.

5.2.1. Continuous binding experiments. CHO hm1 cells

were incubated with a ﬁxed concentration of [³H]NMS

(ranging from 0.14 to 0.20 nM) in the presence of

increasing amounts of the analogs (1–0.1 mM). Non-

speciﬁc binding was deﬁned as residual binding in the

presence of 10 lM atropine. Incubation proceeded for

1 h at 37 ꢁC before termination by rapid ﬁltration

through Whatman GF/B ﬁlters (Whatman, Clifton,

NJ), positioned on a Brandell cell harvester (Gaithers-

burg, MD). Filters were washed three times with 4 mL

of ice-cold saline and dried. Radioactivity was measured

via liquid scintillation counting. Assuming simple com-

petition, the data were reﬁtted according to both one-

and two-site mass-action binding models, and the better

model was determined by an extra-sum-of-squares test

using Prism 3.0 (Prism 3.0, GraphPad, San Diego, CA).

17. Freeman, F.; Kim, D. S.; Rodriguez, E. J. Org. Chem.

1992, 57, 1722–1727.

18. Houssin, R.; Bernier, J. L.; Henichart, J. Synthesis 1988, 3,

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19. Kokotos, G. Synthesis 1990, 4, 299–301.

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22. Kreye, P.; Kihlberg, J. Tetrahedron Lett. 1999, 40, 6113–

6116.

23. McElvain, S. M.; Vozza, J. F. J. Am. Chem. Soc. 1949, 71,

896–897.

5.2.2. Wash-resistant binding experiments. CHO hm1

cells were incubated with xanomeline or its analogs in

the absence of the radioligand (1 h). The incubation

mixture was diluted with ꢀ50 vol of buﬀer and centri-

fuged. The resulting pellet was subjected to this proce-

dure two additional times. The cells were resuspended

in buﬀer and radioligand ([³H]NMS) was added. Incu-

bation proceeded for 1 h at 37 ꢁC before termination

via methods similar to those described in the continuous

binding experiments.

24. Egbertson, M. S.; Chang, C. T.; Duggan, M. E.; Gould, R.

J.; Halczenko, W.; Hartman, G. D.; Laswell, W. L.;

Lynch, J. J., Jr.; Lynch, R. J.; Manno, P. D., et al. J. Med.

Chem. 1994, 37, 2537–2551.

25. Banﬁ, L.; Guanti, G.; Riva, R. Tetrahedron Assym. 1999,

10, 3571–3592.

Acknowledgment

26. Jeppesen, L.; Sauerberg, P. Preparation of aza-

bicycloalkylthiadiazoles as muscarinic agonists. U.S. Pat-

ent 6,015,813, 1998.

This work was supported by NIH Grant NS25743.

27. Sauerberg, P.; Olesen, P. H. Preparation of thiadiazolyl-

substituted azabicyclic compounds for treatment of Alz-

heimer’s disease. U.S. Patent 5,527,813, 1994.

28. Matsui, H.; Lazareno, S.; Birdsall, N. J. Mol. Pharmacol.

1995, 47, 88–98.

29. Spalding, T. A.; Trotter, C.; Skjaerbaek, N.; Messier, T.

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Article Doi

Synthesis and evaluation of xanomeline analogs-Probing the wash-resistant phenomenon at the M1 muscarinic acetylcholine receptor

DOI: 10.1016/j.bmc.2007.10.058

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