1-Benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diols as novel affinity and photoaffinity probes for β-adrenoceptor subtypes

Bioorganic & Medicinal Chemistry 14 (2006) 1684–1697

1-Benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diols as novel

aﬃnity and photoaﬃnity probes for b-adrenoceptor subtypes

Victor I. Nikulin,^aIgor M. Rakov,^aJoseph E. De Los Angeles,^aRatna C. Mehta,^b

LeNe’Sheya Y. Boyd,^cDennis R. Feller^b,cand Duane D. Miller^a,*

^aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee-Memphis, Memphis, TN 38163, USA

^bDivisions of Medicinal Chemistry and Pharmacognosy and Pharmacology, College of Pharmacy,

The Ohio State University, Columbus, OH 43210, USA

^cDepartment of Pharmacology, Research Institute of Pharmaceutical Sciences, National Center for Natural Products Research,

School of Pharmacy, University of Mississippi, University, MS 38677, USA

Received 22 February 2005; accepted 22 July 2005

Available online 20 January 2006

Abstract—Trimetoquinol (TMQ, 1) is a potent non-selective b-adrenoceptor (b-AR) agonist possessing a tetrahydroisoquinoline

(THI) structure. The binding site for 1-trimethoxybenzyl group of 1, which distinguishes it from classical catecholamines, is

unknown. Aﬃnity and photoaﬃnity labeled compounds are good tools to determine the exact interaction between a ligand and

a speciﬁc amino acid(s) in a receptor. In this study, we designed and synthesized a series of aﬃnity 6, 12, 18, and photoaﬃnity

24, 29 labeled analogues of TMQ. All of these compounds were full agonists and demonstrated an equal or greater binding aﬃnity

and functional activity as compared to TMQ on b₁-, b₂-, and b₃-AR. Washout experiments on Chinese hamster ovary (CHO) cells

expressing hu b₂-AR were helpful in identifying the isothiocyanate 18 and the azide 24 as very eﬀective aﬃnity and photoaﬃnity

labels at this receptor subtype.

HO

1. Introduction

NH

Trimetoquinol (TMQ, 1, Chart 1) is a potent b-adreno-

ceptor (b-AR) agonist. The S(ꢀ) isomer of TMQ is cur-

rently used in Japan as a bronchodilatory agent.¹The

3⁰,5⁰-diiodo derivative of TMQ compound 2 is a partial

agonist on hu b₂-AR and a full agonist on hu b₁- and hu

b₃-AR.²The binding aﬃnities of 2 have been reported to

be greater than those of 1.³Like catecholamine adreno-

ceptor agonists such as norepinephrine (NE) and isopro-

terenol (ISO), the structure of TMQ contains both

a catechol and a basic amino group, which comprise

the pharmacophore of this structural class of b-AR

agonists. Several unique structural features diﬀerentiate

TMQ from other typical catecholamine b-AR agonists.

Unlike ﬂexible structures of NE and ISO, the catechol-

amine pharmacophore of TMQ is incorporated in a

rigid THI ring system. Another distinguishing feature

OMe

I

OMe

I

2

TMQ,1

Chart 1.

of TMQ is the lack of a b-hydroxyl group, a substituent

necessary for the potent stereoselective b-AR agonist

activity of NE and ISO.⁴Despite the absence of a b-

OH group, TMQ and related analogues exhibit potent

b-AR activity. The trimethoxy-substituted benzyl group

at the one position of TMQ is a more obvious discerning

structural characteristic that distinguishes TMQ ana-

logues from other catecholamines. This substituted ben-

zyl group is a requirement for the potent b-AR activity

of TMQ and structurally related analogues. Further-

more, the 1-benzyl substituent introduces a chiral center

that diﬀers from those of NE and ISO. Like NE and

ISO, the b-AR agonist action of TMQ is highly

Keywords: b₃-Adrenergic receptor; Agonist; 1,2,3,4-Tetrahydroiso-

quinoline; Photoaﬃnity probe.

*

Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448

6828; e-mail: dmiller@utmem.edu

doi:10.1016/j.bmc.2005.12.027

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1685

stereoselective (S ꢁ R). The structural similarities and

diﬀerences between TMQ and other catecholamines sug-

gest that they interact with the same receptor binding

site domains but diﬀer in their speciﬁc binding interac-

tions within the binding site area.

taining [¹²⁵I]. However, the search for a labeled amino

acid could be performed without a radioactive label

using electrospray ionization mass spectrometry.²¹

2. Chemistry

The b-ARs are classiﬁed into b₁, b₂,⁵and b₃subtypes.^6,7

The b-AR subtypes obtained from diﬀerent species were

cloned and expressed as recombinants in cell lines

including Chinese hamster ovary (CHO) cells. Tradi-

tionally, the therapeutic usefulness of b-AR drugs has

been limited to cardiovascular (b₁-AR) and bronchore-

laxant (b₂-AR) applications. Activation of the b₃-AR

leads to lipolysis in white adipocytes, and both lipolysis

and thermogenesis in brown adipocytes. Moreover, b₃-

AR selective agonists decrease body fat and improve

insulin sensitivity in animal models.^8,9These studies sug-

gest that b₃-AR selective agonists are promising candi-

dates to manage human obesity and type II diabetes.

Also they may be useful for the treatment of gastrointes-

tinal hypermotility disorders.¹⁰

Chloroacetamide 6 was prepared starting from com-

pound 3, in which the synthesis was developed by our

laboratory earlier (Scheme 1).²²Treatment of isoquino-

line 3 with chloroacetyl chloride in the presence of

CaCO₃and 4-(dimethylamino)pyridine (DMAP) result-

ed in chloroacetylation of diiodoaniline moiety. Triﬂuo-

roacetyl (TFA) protection in 4 was removed by mild

basic hydrolysis using K₂CO₃. The resulting dimethoxy

derivative 5 was demethylated with BBr₃to aﬀord the

desired chloroacetamide 6 in 58% the overall yield.

The bromoacetamido-labeling compound 12 was ob-

tained using a single deprotection reagent approach to

reduce the number of deprotection steps (Scheme 1).

This approach required tert-butoxycarbonyl (Boc) pro-

tection of nitrogen in starting nitroisoquinoline 7²²with

Boc₂O and 1 N NaOH. The nitro group in the resulting

compound 8 was reduced with hydrogen over palladium

on activated carbon to give aniline 9, which in turn was

diiodinated with an excess of benzyltrimethylammoni-

um dichloroiodate (BTMAICl₂) in the presence of

CaCO₃according to the procedure by Kajigaeshi

et al.²³Acylation of 10 with bromoacetyl bromide in

the presence of CaCO₃gave triprotected intermediate

11. The latter was deprotected in just one step using

BBr₃to form the desired bromoacetamide analogue 12

in 18% total yield.

b-ARs belong to the superfamily of G-protein-coupled

receptors and have seven transmembrane (TM)-span-

ning domains. TM regions are well conserved among

the three b-AR subtypes. It is assumed that Asp¹¹³in

the TM3 of the b₂-AR forms an ionic bond with the

amino group of ISO,¹¹whereas catechol hydroxyl

groups form hydrogen bonds with Ser²⁰⁴and Ser²⁰⁷in

TM5.¹²We suggest that the same interactions exist for

the TMQ analogues. However, the binding site for the

1-benzyl substituent of TMQ analogues is unknown,

and therefore, the goal of this work is the preparation

of analogues containing aﬃnity labels in the 1-benzyl

group. Labeling experiments on pure cloned receptor

subtypes will give us an opportunity to determine the

nature of the amino acid(s) that interact with the 1-ben-

zyl substituent. This in turn will allow for an improved

model and understanding of ligand–receptor interaction

and might lead to creation of highly selective b-AR ago-

nists with the ultimate goal of ﬁnding potent selective

b₃-AR drugs.

We also attempted to synthesize a monoiodo analogue

of 12. N-Boc-diBnO-protected tetrahydroisoquinoline

13²²was acylated with bromoacetyl bromide in the pres-

ence of triethylamine to give compound 14. It should be

noted that o-iodoacetanilides (like 14) behave as normal

aromatic amides, while di-ortho-substituted acetanilides

(e.g., 11) have tendency to form diacetanilides and one

should be very cautious to make monoacylated prod-

ucts. Similar ortho eﬀect in acetanilides was studied

and explained by Ayyangar and Srinivasan.²⁴Second,

di-ortho-substituted acetanilides are stable toward

deprotection protocols with BBr₃. In contrast, the o-

monoiodoacetanilides are cleaved with BBr₃.²²Taking

all these observations into consideration we tried depro-

tecting isoquinoline 14 with a milder reagent—namely

trimethylsilyl iodide (TMSI). Unfortunately, we isolated

only acetanilide 15—the product of reduction of the

bromoacetamido group.

A variety of aﬃnity and photoaﬃnity b-AR antagonist

analogues have been developed.^13–18Most of these la-

bels identify receptor proteins of M_r58,000 (b₂-subtype)

and of M_r39,000 and M_r45,000 (b₁-subtype). A photo-

aﬃnity analogue of NE labeled a protein band of M_r

65,000 in a guinea pig lung (b₂-subtype), which was

identical to that which was observed with the antagonist

photolabel [¹²⁵I]iodoazidobenzylpindolol.¹⁶Many of

these studies employ mammalian tissues, which contain

diﬀerent ratios of b-subtypes, exhibit high background

binding that may be dependent upon guanine nucleo-

tides. Several photolabels of competitive antagonists

(prenalterol, alprenolol, and CGP 12177) and agonist

(NE) have been successfully used for the derivatization

and isolation of recombinant hamster b₂-AR present

in turkey erythrocytes and guinea pig lung mem-

branes.^19,20To overcome these problems, we continue

to use CHO clones that express homogeneous popula-

tions of human b-AR subtypes. Our new procedures

can be easily applied to the synthesis of analogues con-

The synthesis of isothiocyanato labeled isoquinoline 18

was started from the above-mentioned compound 9

(Scheme 2). Monoiodination of the latter compound

with 1 equiv of BTMAICl₂in the presence of CaCO₃

led to 16, which was treated with thiophosgene in bi-

phase media containing NaHCO₃to obtain isothiocya-

nate 17.²⁵Standard deprotection of 17 with BBr₃

followed by recrystallization from cold methanol–ether

gave the ﬁnal isothiocyanate 18 with a 61% overall yield.

1686

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

MeO

ClCOCH Cl

2

K CO

2

3

NTFA

MeO

CaCO , DMAP

3

I

NH

NHCOCH Cl

2

4

3

I

MeO

HO

BBr

3

NH

NH • HBr

I

NHCOCH Cl

2

6

5

I

MeO

(Boc) O

2

BTMAICl

H , Pd/C

2

NBoc

NH

NaOH

CaCO

3

NH

NO

9

8

NO

7

2

HO

MeO

BrCOCH Br

BBr

2

3

NH • HBr

I

NBoc

CaCO

3

I

NHCOCH Br

NH

2

NHCOCH Br

2

11

12

I

10

I

HO

BnO

TMSI

BrCOCH Br

2

NH • HI

NBoc

Et N

3

I

NHCOCH

NHCOCH Br

NH

3

2

15

14

13

Scheme 1.

MeO

CSCl , CHCl

BTMAICl

2

3

2

NBoc

CaCO

3

NaHCO , H O

I

3

2

NH

2

16

9

2

MeO

HO

BBr

3

NBoc

NH • HBr

I

NCS

17

NCS

18

Scheme 2.

The most challenging project was the synthesis of azide

24. The diﬃculties arise from lability of the azido group

toward acids and Lewis acids and decomposition of

catechol moiety in alkaline solutions. The synthesis

of a linear catecholamine azide was described by

Ruoho et al.^16,19The strategy for the synthesis of

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1687

cyclic azidocatecholamines like tetrahydroisoquinolines

should be diﬀerent considering the very strenuous condi-

tions of Bischler–Napieralski cyclization. We decided to

reprotect the catechol fragment with carbonate groups

and protect isoquinoline nitrogen with 9-ﬂuorenyl-

methoxycarbonyl (Fmoc) group (Scheme 3). This strat-

egy allowed us to use a basic amine as a single

deprotecting reagent. Indeed, isoquinoline 7 was pro-

tected with Fmoc-Cl in the presence of N,N-diisopropyl-

ethylamine (DIPEA) to give derivative 19, which was

demethylated with BBr₃. Acylation of the resulting cat-

echol 20 with ethyl chloroformate aﬀorded triprotected

analogue 21. The nitro group in the latter compound

was reduced with hydrogen in the presence of palladium

on activated charcoal followed by monoiodination with

1.1 equiv of BTMAICl₂in the presence of CaCO₃to

give o-iodoaniline 22. A treatment of the diazonium salt,

formed by the addition of aqueous solution of NaNO₂

to an acetic acid solution of compound 22, with a

solution of NaN₃led to a protected azide 23. The latter

was treated with pyrrolidine, puriﬁed by ﬂash chroma-

tography, and the desired iodoazide 24 was isolated as

HCl salt in 23% overall yield.

4-Benzoylphenylacetic acid 25 for the synthesis of ben-

zophenone label 29 was obtained in three steps from

p-methylbenzophenone according to the procedure by

Zderic et al.²⁶Acid 25 was transformed into acid chlo-

ride by heating with SOCl₂followed by reaction with

3,4-dimethoxyphenethylamine in the presence of trieth-

ylamine (Scheme 4). The carbonyl group in the resulting

amide 25 was protected with 1,2-ethanedithiol using

BF₃ÆEt₂O as a catalyst.²⁷Bischler–Napieralski cycliza-

tion of the resulting oily product in POCl₃/MeCN and

reduction with NaBH₄in methanol led to compound

27. The dithiolane ring in compound 27 was smoothly

cleaved with mercury (II) perchlorate to form ketone

28.²⁷The desired benzophenone containing photo-

aﬃnity label 29 was obtained after standard demethyla-

tion of compound 28 with BBr₃in 31% total yield.

MeO

HO

MeO

EtOCOCl

Py

BBr

Fmoc-Cl

3

N-Fmoc

NH

HO

MeO

DIPEA

7

20

19

NO

2

NO

2

EtOCO

2

EtOCO

2

1. NaNO , AcOH

2

1. H , Pd/C

2

N-Fmoc

I

N-Fmoc

2

2. NaN

3

2

2. BTMAICl

2

22

NH

21

2

NO

2

EtOCO

HO

NH

2. HCl

2

1.

N-Fmoc

NH • HCl

I

2

I

24

23

N

3

N

3

Scheme 3.

O

H

N

1. SOCl

MeO

1. (HSCH ) , BF •Et O

2

2 2

3

2

O

MeO

NH

2

COOH

2. POCl

3

2.

3. NaBH

4

MeO

26

O

25

Et N

3

MeO

HO

MeO

Hg(ClO )

BBr

3

NH

NH • HBr

4 2

NH

S

O

29

28

27

Scheme 4.

1688

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

It should be noted that the ¹H and ¹³C NMR spectra of

N-Boc and N-Fmoc derivatives are quite complicated

displaying signals corresponding to two major rotamers.

3. Biological results and discussion

One objective of this research is to examine whether

appropriately modiﬁed 1-benzyl TMQ analogues are

suitable as aﬃnity probes for b-AR subtypes. Biochem-

ical and functional characterization of aﬃnity labels 6,

12, and 18 on rat and human b₃-AR was discussed in

our article recently.²⁸Therefore, it was desirable to char-

acterize the aﬃnity and photoaﬃnity characteristics of

the compounds we synthesized in CHO cells expressing

the hu b₂-AR. 1-(4-Acetamido-3,5-diiodobenzyl) deriva-

tive of TMQ 30, synthesized earlier,²²was used for

comparison.

Table 1 compares the binding aﬃnities and functional

activities of new analogues at human b₁-, b₂-, or b₃-

AR to those of the reference compounds, ISO and

TMQ. Both cAMP radioimmunoassay (cAMP-RIA)

and cAMP response element-luciferase reporter gene

(CRE-LUC) assay were employed to determine the

receptor activities of the compounds. The non-selective

b-AR agonist, (ꢀ)ISO, and 1-benzyl ring-substituted

derivatives of TMQ competed for speciﬁc-bound [¹²⁵I]-

(ꢀ)-3-iodocyanopindolol (ICYP) from the hu b-AR in

a concentration-dependent manner. In studies with hu

b₁- and hu b₂-ARs, the non-speciﬁc [¹²⁵I]ICYP binding

was less than 5%, whereas with hu b₃-AR studies,

non-speciﬁc binding was up to 30% of the total binding.

At high concentrations, these ligands completely inhibit-

ed speciﬁc binding of ICYP to the hu b₁- and hu

b₂-ARs, whereas in the hu b₃-AR system, only 60–90%

of the speciﬁc binding of ICYP was inhibited by most

TMQ analogues, an exception being azide 24 which

completely abolished ICYP binding to this receptor sub-

type at high concentrations. In general, the compounds

including ISO exhibited similar binding aﬃnities for

both hu b₁- and hu b₂-ARs. However, the TMQ deriva-

tives possessed signiﬁcantly higher (10- to 220-fold)

binding aﬃnities for the b-AR as compared to ISO.

Predictably, all the ligands exhibited signiﬁcantly lower

aﬃnities for the hu b₃-AR as compared to the other

two subtypes. Typically, the compounds showed 20- to

50-fold lower aﬃnities for the hu b₃-AR subtype, with

the exception of the azido derivative 24 which exhibited

only 3- to 6-fold diﬀerence in binding aﬃnities for the

b-AR subtypes. The pK_is for azide 24 on all b-AR

subtypes were over 6.70, which are greater than pK_is

of 4–6 required for phenyl azides.¹⁹

Biochemical potencies expressed as pK_aindicated that

all the TMQ analogues were more potent than ISO in

CHO cells expressing all subtypes of hu b-ARs, the azide

24 being the most active in all cases, reaching the 13 pM

level at b₂-AR. Also, the maximal intrinsic activities of

the aﬃnity and photoaﬃnity labels were 0.94–1.42 as

compared to ISO (1.0), indicating these compounds to

be full agonists in the hu b-AR systems.

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1689

3.1. Aﬃnity labeling

binding. Non-speciﬁc radioligand binding to the recep-

tors was around 5%. Thus, compound 8 was a more

eﬀective aﬃnity ligand on the hu b₂-AR, whereas 6

exhibited weak irreversibly acting aﬃnity properties on

this system.

In reversible competitive radioligand binding experi-

ments, all of the selected 1-benzyl-substituted analogues

of TMQ had inhibited [¹²⁵I]ICYP binding to the hu b₂-

AR in CHO cells in a concentration-dependent manner

(Table 1). In washout experiments, concentration-de-

pendent inhibition of [¹²⁵I]ICYP binding to hu b₂-AR

was observed in cells pretreated with isothiocyanate 18

or chloroacetamide 6. In contrast, acetamide 30 pre-

treatment did not signiﬁcantly aﬀect radioligand binding

after washout, irrespective of the concentration used

(Fig. 1). Depending upon the concentration used, isothi-

ocyanate 18 inhibited ICYP binding by 35–85%,

whereas chloroacetamide 6 inhibited ICYP binding by

a maximum of only 30–40% of the total radioligand

Figure 2 shows the eﬀect of incubating acetamide 30,

chloroacetamide 6, and isothiocyanate 18 over a range

of 2–45 min at various concentrations with hu b₂-AR-

CHO in these washout experiments. At concentrations

up to 30K_i, the irreversible binding of all the three com-

pounds to the receptor appeared to improve with incu-

bation periods of up to 15 min, while slight recovery in

ICYP binding occurred at an incubation period of

45 min. However, this trend was abolished when the

compounds were incubated at higher concentrations

100

80

100

80

60

Acetamide 30

40

20

0

20

0

15

30

45

3 K_i

10 K_i

30 K_i100 K_i

100

80

100

80

60

40

20

0

60

40

Chlorocetamide 6

Chloroacetamide 6

20

0

15

30

10 K_i

30 K_i100 K_i

100

80

60

40

20

0

Isothiocyanate 18

80

60

40

20

0

Isothiocyanate 18

15

30

10 K_i

30 K_i100 K_i

Incubation Time (min)

[Drug] (M)

Figure 2. Eﬀect of time of incubation with ligands at molar concen-

tration of 3K_i(d), 10K_i(s), 30K_i(.) and 100K_i(,) in washout

experiments, upon the inhibition of [¹²⁵I]ICYP binding to hu b₂-AR in

CHO cells. The data are means SEM of three experiments, each in

triplicate.

Figure 1. Concentration-dependent inhibition of [¹²⁵I]ICYP binding to

hu b₂-AR in CHO cells in washout experiments after incubation with

selected TMQ analogues for 2 min (,), 5 min (d), 15 min (s) or 45 min

(.). Values are means SEM of three experiments, each in triplicate.

1690

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

(100-fold of their K_ivalues) over this time range. Thus,

there seems to be a more distinctive concentration

dependency relationship for the irreversible binding of

isothiocyanate 18 and chloroacetamide 6 analogues as

compared to the relationship of binding to the period

of incubation. In the case of the isothiocyanato 18 ana-

logue, although a slightly greater eﬀect was observed

upon incubation for 15 min, shorter time periods of 2

and 5 min were suﬃcient to demonstrate ‘irreversible’

binding of the compound to the hu b₂-AR. Therefore,

the binding by analogue 18 exhibits very rapid reaction

kinetics. Collectively, the concentration and time data

indicate that isothiocyanate 18 is an eﬀective aﬃnity

analogue on the hu b₂-AR.

ligand, the radioligand binding of ICYP was not signif-

icantly aﬀected (Fig. 3). Similarly, insigniﬁcant diﬀerenc-

es in the extent of ICYP binding were observed between

samples incubated in complete dark environment and

those under red light for 1 h.

4. Conclusions

We have developed the synthesis and evaluated the

interaction of aﬃnity and photoaﬃnity labels of the

TMQ class for b-AR. Each compound showed high

binding aﬃnity and potency, and the compounds were

full agonists on the three subtypes of human b-AR.

Washout studies on b₂-AR demonstrated that isothiocy-

anate 18 reacted quickly to produce up to 85% of irre-

versible binding to the receptor subtype. Photoaﬃnity

binding with the azide 24 reached up to 87% at the same

receptor. Thus in this study, we have discovered one

eﬀective aﬃnity agonist label and one photoaﬃnity ago-

nist label for the b₂-AR belonging to the 1-benzyl-

1,2,3,4-tetrahydroisoquinoline-6,7-diol chemical class.

3.2. Photoaﬃnity labeling

When CHO cells expressing the hu b₂-AR were incubat-

ed with various concentrations of azide 24 under a red

light for 1 h and exposed to UV light for a ﬁxed amount

of time (0.5 and 5 min), a concentration-dependent inhi-

bition of ICYP binding to these cells was observed. This

ﬁnding indicated that the azido analogue 24 was photo-

lyzable and bound irreversibly to this receptor (see

Fig. 3). At higher concentrations (10- and 100K_i) of

the photoaﬃnity ligand 24, inhibition of ICYP binding

reached 47–87% of the total binding, and was dependent

upon the sample exposure times to UV light of 350 nm.

The extent of photoaﬃnity ligand irreversible binding

was directly proportional to its concentration and time

period of exposure to UV light. Samples incubated with

the azido analogue 24 under the red light, but not ex-

posed to UV light, did not exhibit irreversible binding

which implies that the exposure of the azido analogue

24 to UV light is necessary for the formation of the high-

ly reactive nitrene species under these photolytic condi-

tions, which covalently binds to the receptor protein.

5. Experimental

5.1. Chemistry

Melting points were determined on a Thomas–Hoover

capillary melting point apparatus and are uncorrected.

Infrared spectra were recorded on a Perkin Elmer Sys-

tem 2000 FT-IR. Proton and Carbon-13 NMR spectra

were obtained on a Bruker AX 300 spectrometer.

Chemical shift values are reported as parts per million

(d) relative to tetramethylsilane (TMS) as an internal

standard. Spectral data are consistent with assigned

structures. Elemental analyses were performed by

Atlantic Microlab Inc., Norcross, GA, and found val-

ues are within 0.4% of the theoretical values. Flash

chromatography was performed on silica gel (Merck,

In control experiments where CHO cells were exposed to

UV light for 5 min in the absence of the photoaﬃnity

˚

grade 60, 230–400 mesh, 60 A). Anhydrous solvents

were purchased from Aldrich.

120

100

5.1.1.

1-(4-a-Chloroacetamido-3,5-diiodobenzyl)-2-tri-

ﬂuoroacetyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline

(4). Chloroacetyl chloride (0.213 g, 1.88 mmol) was

added to a cooled (0 ꢁC) and stirred solution of 3

(0.44 g, 0.68 mmol), CaCO₃(0.7 g, 6.99 mmol), and

DMAP (1 mg) in dry CH₂Cl₂(40 mL). The mixture

was allowed to stir at room temperature overnight

(15 h). Satd NaHCO₃(20 mL) was added to the solution

and the mixture was stirred for 1 h. The mixture was

extracted with CH₂Cl₂(3· 50 mL). The organic extract

was dried over Na₂SO₄then evaporated in vacuum to

give a white solid. Recrystallization from ethyl ace-

tate–hexanes mixture gave 0.37 g (69%) of the product

0 min

80

60

40

30 s

20

5 min

0

1

10 K_i

3 K_i

[Drug] (M)

1 K_i

100 K_i

as a white solid: mp 261–263 ꢁC; H NMR (300 MHz,

CDCl₃) d 8.09 (s, 1H, CONH), 7.65 (br m, 2H, ArH),

6.63 (s, 1H, ArH), 6.26 (s, 1H, ArH), 5.48 (t, 1H,

CH), 4.25 (s, 2H, CH₂), 3.94–3.98 (m, 1H, CH), 3.87

(s, 3H, CH₃), 3.74 (s, 3H, CH₃), 3.64–3.67 (m, 1H,

CH), 2.90–3.05 (m, 3H, CH), 2.65–2.80 (m, 1H, CH);

Figure 3. Concentration-dependent inhibition of [¹²⁵I]ICYP binding to

hu b₂-AR in CHO cells in washout experiments by the azide 24 after

incubation in red light for 1 h followed by exposure to UV light at

350 nm for 0 min, 30 s or 5 min. The data are means SEM of two–six

experiments, each in triplicate.

IR (KBr) 3434 (NH), 1720 (C@O), 1684 (C@O) cm^ꢀ1

.

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1691

Elemental analysis

Compound

Formula

Calculated (%)

Found (%)

H

C

H

N

C

N

6

9

C₁₈H₁₇ClI₂N₂O₃ÆHBrÆ0.15 Et₂O

C₂₃H₃₀N₂O₄

C₂₃H₂₈I₂N₂O₄Æ0.4 C₆H₁₄

C₁₈H₁₈N₂O₃Br₂I₂Æ0.33 Et₂O

C₃₇H₃₈BrIN₂O₅

C₁₈H₁₉IN₂O₃ÆHIÆ0.5 EtOAc

C₂₃H₂₉IN₂O₄

C₂₄H₂₇IN₂O₄SÆ0.1 CHCl₃

C₁₇H₁₅IN₂O₂SÆHBr

C₃₃H₃₀N₂O₆Æ0.45 CH₂Cl₂

C₃₁H₂₆N₂O₆

C₃₇H₃₄N₂O₁₀

C₃₇H₃₅IN₂O₈

C₃₇H₃₃IN₄O₈

C₁₆H₁₅IN₄O₂ÆHClÆ0.5 Et₂O

C₂₅H₂₅NO₄Æ0.05 CH₂Cl₂

C₂₇H₂₉NO₂S₂Æ(COOH)₂

C₂₅H₂₅NO₃Æ(COOH)₂

C₂₃H₂₁NO₃ÆHBr

32.35

69.32

44.55

31.02

55.72

39.37

52.68

50.05

39.33

68.23

71.25

66.66

58.27

56.35

43.61

73.79

62.91

67.91

62.74

2.85

7.59

4.95

2.87

4.80

3.96

5.57

4.72

3.11

5.29

5.02

5.14

4.63

4.22

4.21

6.21

5.64

5.70

5.04

4.06

32.34

69.21

44.48

31.15

55.65

39.55

52.61

49.92

39.40

68.20

70.98

66.53

58.32

56.21

43.51

73.75

62.81

67.68

62.48

2.84

7.61

4.87

2.80

4.81

4.03

5.56

4.70

3.14

5.40

5.10

5.20

4.67

4.18

4.21

6.30

5.71

5.78

5.08

4.05

7.03

4.09

3.74

3.51

4.59

5.34

4.84

5.40

4.76

5.36

4.20

3.67

7.10

11.30

3.44

2.53

2.93

3.18

7.05

4.08

3.68

3.52

4.69

5.25

4.78

5.31

4.66

5.27

4.14

3.64

7.04

11.09

3.43

2.49

2.88

3.05

10

12

14

15

16

17

18

19

20

21

22

23

24

26

27

28

29

5.1.2.

1-(4-a-Chloroacetamido-3,5-diiodobenzyl)-6,7-

(s, 1H, ArH), 6.72 (s, 1H, ArH), 6.56 (s, 1H, ArH),

4.66 (br m, 1H, CH), 4.27 (s, 2H, CH2), 3.3–3.38 (m,

2H, CH), 3.10–3.20 (m, 2H, CH), 2.80–3.0 (m, 4H,

dimethoxy-1,2,3,4-tetrahydroisoquinoline (5). A solution

of K₂CO₃(1.4 g, 10 mmol) in 1:1 methanol/H₂O

(40 mL) was added to a solution of 4 (0.37 g, 0.5 mmol)

in methanol (30 mL). The mixture was stirred until com-

pletely dissolved (ꢂ7 h). The solution was concentrated

in vacuum and extracted with ethyl acetate (3· 75 mL).

The organic extract was dried with Na₂SO₄and evapo-

rated in vacuum to give 0.24 g (93%) of the product as a

CH); IR (KBr) 1684 (C@O), 1518 (C@C, Ar) cm^ꢀ1

Anal. (C₁₈H₁₇ClI₂N₂O₃ÆHBrÆ0.15 Et₂O) C, H, N.

.

5.1.4. 2-tert-Butoxycarbonyl-6,7-dimethoxy-1-(4-nitro-

benzyl)-1,2,3,4-tetrahydroisoquinoline (8). To a cooled

(0 ꢁC) and stirred solution of 7 (2.0 g, 6.09 mmol) in

THF (50 mL) was added 1 N NaOH (15 mL). Di-tert-

butylcarbonate (1.33 g, 6.09 mmol) was added to the

solution and stirring was continued for 30 min at 0 ꢁC.

The reaction mixture was stirred at room temperature

for 24 h. The organic solvent was stripped in vacuum

and the aqueous residue was diluted with water

(20 mL). The cloudy solution was extracted with

CH₂Cl₂(3· 75 mL). The organic extract was dried

(Na₂SO₄), then evaporated to give a glassy solid.

Recrystallization from CH₂Cl₂–hexanes mixture gave

2.14 g (82%) of the product as light yellow crystals:

mp 137–139 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 6.87

(m, 2H, ArH), 6.6 (m, 3H, ArH), 6.33 (s, 1H, ArH),

5.1 (t, J = 6.8 Hz, 1H), 4.15 (m, 1H, CH), 3.85 (s, 3H,

OMe), 3.73 (s, 2H, Me), 3.64 (s, 1H, OMe), 3.25 (m,

1H, CH), 2.5–3.05 (m, 4H, CH), 1.45 (s, 3H, Me), 1.35

(s, 6H, Me); IR (KBr) 1682 (C@O) cm^ꢀ1; MS m/e: 428

(M⁺, EI).

1

beige solid: mp 255 ꢁC (dec.); H NMR (300 MHz) d

8.11 (s, 1H, CONH), 7.82 (s, 1H, ArH), 7.26 (s, 1H,

ArH), 6.60 (s, 1H, ArH), 6.58 (s, 1H, ArH), 4.27 (s,

2H, CH₂Cl), 4.14 (m, 1H, CH), 3.86 (s, 3H, OMe),

3.84 (s, 3H, OMe) 3.08–3.17 (m, 2H, CH), 2.97 (m,

1H, CH), 2.7–2.84 (m, 2H, CH), 1.85 (br s, 1H, NH);

IR (KBr) 1684 (C@O), 1518 (C@C, Ar) cm^ꢀ1

.

5.1.3. 1-(4-a-Chloroacetamido-3,5-diiodobenzyl)-6,7-di-

hydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide

(6). To a solution of 5 (0.523 g, 0.83 mmol) in dry

CH₂Cl₂(50 mL) at ꢀ78 ꢁC was added dropwise 4 mL

of 1 M solution of BBr₃in CH₂Cl₂under an argon

atmosphere. The reaction mixture was allowed to warm

to room temperature and stirring was continued over-

night. The mixture was cooled (ꢀ78 ꢁC) and quenched

with methanol (20 mL). Stirring was continued at room

temperature for 2 h. The solvents were evaporated in

vacuum. A residue was dissolved in methanol (50 mL)

and the solvent was removed in vacuum. This procedure

was repeated four times to give a solid residue. Recrys-

tallization from methanol–ethyl ether mixture gave

0.51 g (90%) of the product as an oﬀ-white solid: mp

202 ꢁC; ¹H NMR (300 MHz, DMSO-d₆) d 10.24 (s,

1H, CONH), 9.15 (br m, 1H, OH), 8.05 (br m, 2H,

NH⁺), 8.54 (br m, 1H, OH), 7.99 (s, 1H, ArH), 7.95

5.1.5. 1-(4-Aminobenzyl)-2-tert-butoxycarbonyl-6,7-di-

methoxy-1,2,3,4-tetrahydroisoquinoline (9). A solution

of 8 (2.0 g, 4.7 mmol) in ethyl acetate (100 mL) was

hydrogenated (50 psi) over 10% Pd/C for 40 min. The

catalyst was removed by ﬁltration and the ﬁltrate was

concentrated to about 1/3 the original volume. Hexane

was added until the solution became slightly cloudy.

1692

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

The solution was allowed to stand at room temperature

overnight. The white crystalline product (1.71 g, 91%)

was collected by ﬁltration: mp 170–172 ꢁC; IR (KBr)

3432, 3353, 3241 (NH), 1668 (C@O) cm^ꢀ1; MS m/e

398 (M⁺, EI). Anal. (C₂₃H₃₀N₂O₄₎C, H, N.

by ﬁltration to give 0.21 g (85%) of the product as an

oﬀ-white powdery solid: mp 210 ꢁC (dec.); ¹H NMR

(300 MHz, DMSO-d₆) d 10.3 (s, 1H, CONH), 9.15 (br m,

1H, OH), 8.95 (br m, 2H, NH⁺), 8.63 (br m, 1H, OH),

7.99 (s, 1H, ArH), 7.96 (s, 1H, ArH), 6.72 (s, 1H, ArH),

6.56 (s, 1H, ArH), 4.68 (br m, 1H, CH), 4.06 (s, 2H,

CH₂), 3.28–3.38 (m, 2H, CH), 3.05–3.16 (m, 2H, CH),

2.70–2.97 (m, 4H, CH); ¹³C NMR (75 MHz, DMSO-d₆)

d 168.03 (C@O), 147.05 (Ar), 145.88 (Ar), 142.04 (Ar),

141.89 (Ar), 141.45 (Ar), 140.40 (Ar), 123.69 (Ar), 123.04

(Ar), 116.31 (Ar), 114.11 (Ar), 100.00 (Ar), 99.99 (Ar),

57.40 (Al), 41.08 (Al), 39.87 (Al), 28.65 (Al), 25.66 (Al);

IR (KBr) 1669 (C@O), 1522 (C@C, Ar) cm^ꢀ1; MS m/e

643, 645 (M+H, M+2+H, FAB). Anal. (C₁₈H₁₈N₂O_3-

Br₂I₂Æ0.33 Et₂O) C, H, N.

5.1.6. 1-(4-Amino-3,5-diiodobenzyl)-2-tert-butoxycarbon-

yl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline

(10).

CaCO₃(2.93 g, 29.3 mmol) and BTMAICl₂(1.33 g,

3.76 mmol) were added to a solution of 9 (1.50 g,

3.76 mmol) in a mixture of CH₂Cl₂(100 mL) and

MeOH (40 mL). The reaction mixture was stirred at

room temperature for 1 h. Additional BTMAICl₂

(3.35 g, 9.4 mmol) was added in two portions over a 2-

day period. The solution was ﬁltered, washed with 5%

Na₂SO₃(150 mL), and dried (MgSO₄). Evaporation of

the solvent gave a glassy light red solid. Recrystalliza-

tion from ethyl acetate–hexanes mixture gave 1.23 g

(50%) of the product as peach colored crystals: mp

112–114 ꢁC; IR (KBr) 3436 (NH), 3350 (NH), 1674

(C@O) cm^ꢀ1. Anal. (C₂₃H₂₈I₂N₂O₄Æ0.4 C₆H₁₄) C, H, N.

5.1.9. 1-(4-a-Bromoacetamido-3-iodobenzyl)-6,7- dibenzyl-

oxy-2-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline

(14). To a cold solution (0 ꢁC) of isoquinoline 13 (0.68 g,

1 mmol) and Et₃N (0.34 g, 3 mmol) in CH₂Cl₂(10 mL)

was added BrCOCH₂Br (0.40 g, 2 mmol). The cooling

bath was removed and the mixture was stirred overnight.

CH₂Cl₂was added and the solution was washed with

water, dried over MgSO₄, ﬁltered, and concentrated.

The resulting solution was puriﬁed by column chroma-

tography (silica gel, hexane/EtOAc, 2:1). The solvents

were evaporated under reduced pressure, ethyl ether

was added, and evaporated to give a white glassy solid

5.1.7. 1-(4-a-Bromoacetamido-3,5-diiodobenzyl)-2-tert-bu-

toxycarbonyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline

(11). A mixture of 10 (0.70 g, 1.1 mmol) and CaCO₃(1 g,

10 mmol) in CH₂Cl₂(14 mL) was cooled to 0 ꢁC in an ice

bath. A solution of bromoacetyl bromide (0.232 g,

1.15 mmol) in CH₂Cl₂(2 mL) was added dropwise with

stirring. After the addition, the reaction mixture was stir-

red at room temperature for 45 min. The mixture was

diluted with CH₂Cl₂(40 mL) and ﬁltered. The ﬁltrate

was washed with saturated NaHCO₃solution (40 mL)

and dried with Na₂SO₄. The solvent was evaporated in

vacuum to give a white solid. Recrystallization of the

crude product from ethyl acetate–hexanes mixture gave

1

(0.44 g, 55%); H NMR (300 MHz, CDCl₃) d (mixture

of conformers of 3:2 ratio) 8.54 (s, 1H, NH), 8.11 and

8.06 (s, 1H, H-5⁰), 7.60–7.25 (m, 11H, 2· Ph+H-2⁰),

7.08 and 7.00 (m, 1H, H-6⁰), 6.70 and 6.66 (s, 1H, H-5),

6.47 and 6.35 (s, 1H, H-8), 5.25–4.88 (m, 2· CH₂O+H-

1), 4.11 and 3.73 (m, 1H, H-3e), 4.05 (m, 2H, CH₂Br),

3.28–3.10 (m, 1H, H-3a), 3.98–2.60 (m, 3H, CH₂

Ar+H-4a), 2.54 and 2.42 (m, 1H, H-4e), 1.43 and 1.31

1

0.49 g (58%) of the product: mp 172–174 ꢁC (dec.); H

NMR (300 MHz, CDCl₃) d 7.94 (s, 1H, NH), 7.64 (br

s, 1H, ArH), 7.59 (br s, 1H, ArH), 6.60 (s, 1H, ArH),

6.28 and 6.23 (s, 1H, ArH), 5.2 and 5.05 (m, 1H, CH),

4.15 (m, 1H, CH), 4.05 (s, 2H, CH₂Br), 3.85 (s, 3H,

OMe), 3.76 and 3.70 (s, 3H, OMe), 3.5–3.4 (m, 1H,

CH), 2.65–3.05 (m, 4H, CH); IR (KBr) 3352 (NH),

(t-Bu); IR (KBr) 1688 (C@O), 1518 (C@C Ar) cm^ꢀ1

.

Anal. (C₃₇H₃₈BrIN₂O₅) C, H, N.

5.1.10.

1-(4-Acetamino-3-iodobenzyl)-6,7-dihydroxy-

1,2,3,4-tetrahydroisoquinoline hydroiodide (15). To a solu-

tion of isoquinoline 14 (0.40 g, 0.5 mmol) in anhydrous

MeCN (5 mL) was added Me₃SiI (0.40 g, 2 mmol) via syr-

inge under an argon atmosphere. The solution was stirred

for 6 h followed by addition of MeOH (1 mL) and stirring

was continued for 10 min. CH₂Cl₂(50 mL) was added to

reaction mixture and yellow crystals were ﬁltered, yield

0.19 g (62%). Analytical sample was obtained by recrys-

tallization from MeOH/MeCN/EtOAc mixture, mp

1687 (C@O) cm^ꢀ1

.

5.1.8. 1-(4-a-Bromoacetamido-3,5-diiodobenzyl)-6,7-dihy-

droxy-1,2,3,4-tetrahydroisoquinoline hydrobromide (12).

A stirred solution of 11 (0.26 g, 0.34 mmol) in anhydrous

CH₂Cl₂(15 mL) was cooled in a dry ice-acetone bath and

kept under an argon atmosphere. A 1 M solution of BBr₃

in CH₂Cl₂(1.3 mL, 1.3 mmol) was slowly added via syr-

inge. The reaction mixture was allowed to warm to room

temperature and stirring was continued overnight. The

mixture was cooled in an ice bath and carefully quenched

with methanol (5 mL). Stirring was continued at room

temperature for 2 h. The solvent was evaporated in vacu-

um and the oily residue was taken up in methanol

(10 mL). The solvent was evaporated in vacuum. The

methanol addition and evaporation were repeated three

times to give an oily residue. The residue was taken up

in a minimum amount of methanol. A small amount of

anhydrous ether was added until formation of a precipi-

tate. The cloudy solution was allowed to stand in a refrig-

erator overnight. The precipitated product was collected

1

172–174 ꢁC (dec.); H NMR (300 MHz, DMSO-d₆) d

9.39 (s, 1H, NH), 8.86 (br s, 1H, OH), 8.50 (br s, 1H,

OH), 7.90 (d, J_m= 1.7 Hz, 1H, H-2⁰), 7.41 (d, J_o= 8.2 Hz,

1H, H-5⁰), 7.35 (dd, J_o= 8.2, J_m= 1.7 Hz, 1H, H-6⁰), 6.63

(s, 1H, H-5), 6.56 (s, 1H, H-8), 4.63 (m, 1H, H-1), 3.43–

2.70 (m, 6H, H-3+H-4+CH₂Ar), 2.06 (s, 3H, Ac); ¹³C

NMR (75 MHz, methanol-d₄) d 172.65 (s, C@O), 146.98

(s), 145.81 (s), 141.48 (d), 140.11 (s), 137.15 (s), 131.30

(d), 129.10 (d), 123.63 (s), 123.27 (s), 116.27 (d), 114.15

(d), 97.83 (s, C-3⁰), 57.60 (d, C-1), 41.01 (t), 39.98 (t),

25.68 (t), 23.09 (q, Me); IR (KBr) 3600–2400 (br, OH,

NH), 1655 (C@O), 1624 (NH bend), 1522 (C@C Ar)

cm^ꢀ1; MS m/e 439 (M⁺). Anal. (C₁₈H₁₉IN₂O₃ÆHIÆ0.5

EtOAc) C, H, N.

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1693

5.1.11. 1-(4-Amino-3-iodobenzyl)-2-tert-butoxycarbonyl-

6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (16). CaCO₃

(1.7 g, 17 mmol) and BTMAICl₂(0.76 g, 2.18 mmol) were

added to a solution of 9 (0.87 g, 2.18 mmol) in a mixture

of CH₂Cl₂(50 mL) and MeOH (20 mL). The reaction mix-

ture was stirred at room temperature for 1 h. The solution

was ﬁltered, washed with 5% Na₂SO₃(100 mL) and dried

over MgSO₄. Evaporation of the solvent gave a glassy light

pink solid, yield 0.98 g (86%): mp 88–90 ꢁC; IR (KBr) 3452

(NH), 3360 (NH), 1684 (C@O) cm^ꢀ1; MS m/e 525 (M+H,

FAB). Anal. (C₂₃H₂₉IN₂O₄) C, H, N.

(1.29 g, 10 mmol) in 10 mL of CH₂Cl₂slowly. The cooling

bath was removed and the solution was stirred for 2 h. The

reaction mixture was washed twice with 1 N HCl, twice

with water, dried over MgSO₄, ﬁltered, concentrated, and

recrystallized from AcOEt–hexanes. Filtration gave

5.67 g (96%) of yellow crystals: mp 140–141 ꢁC; ¹H

NMR (300 MHz, CDCl₃) d (mixture of conformers of

10:9 ratio) 8.07 (d, J = 8.5 Hz, ArH), 7.97 (d, J = 8.4 Hz,

ArH), 7.78 (d, J = 7.5 Hz, ArH), 7.73 (d, J = 7.4 Hz,

ArH), 7.61 (d, J = 7.3 Hz, ArH), 7.56 (d, J = 7.3 Hz,

ArH), 7.17–7.47 (m, ArH), 6.80 (d, J = 8.4 Hz, ArH),

6.60 (s, ArH), 6.55 (s, ArH), 6.29 (s, ArH), 6.02 (s, ArH),

5.32 (t, J = 6.5 Hz, CH), 4.73 (dd, J = 10.9, 4.8 Hz, CH),

4.64 (t, J = 6.8 Hz, CH), 4.36–4.50 (m, CH), 4.22 (t,

J = 6.6 Hz, CH), 3.82–3.97 (m, CH), 3.86 (s, OMe), 3.70–

3.80 (m, CH), 3.69 (s, OMe), 3.07–3.37 (m, CH), 2.61–

2.88 (m, CH), 2.42–2.57 (m, CH); ¹³C NMR (75 MHz,

CDCl₃) d (conformers’ mixture) 155.38 and 155.11

(C@O), 148.06, 147.32, 147.02, 146.73, 146.56, 145.94,

145.69, 144.07, 143.89, 143.88, 143.58, 141.48, 141.34,

130.55, 130.36, 127.71, 127.52, 127.41, 127.18,

127.01,126.53, 126.29, 124.87, 124.81, 124.59, 123.30,

123.20, 119.99, 119.83, 111.39, 111.29, 110.11, 109.93,

67.29 and 66.08 (OCH₂), 55.87 (OMe), 55.66 (C-1), 47.72

and 47.28 (CHAr₂), 42.50 and 42.42 (C-3), 39.40 and

38.59 (CH₂Ar), 27.94 and 27.50 (C-4); IR (KBr) 1688

(C@O), 1607, 1520 and 1347 (NO₂) cm^ꢀ1. Anal.

(C₃₃H₃₀N₂O₆Æ0.45 CH₂Cl₂) C, H, N.

5.1.12.

2-(tert-Butoxycarbonyl)-1-(3-iodo-4-isothio-

cyanatobenzyl)-6,7-dimethoxy-1, 2,3,4-tetrahydroisoquin-

oline (17). To a mixture of isoquinoline 16 (0.52 g,

1 mmol) in CHCl₃(10 mL) and NaHCO₃(0.84 g,

10 mmol) in H₂O (10 mL) was added CSCl₂(0.23 g,

2 mmol). The mixture was stirred at room temperature

for 1 h. H₂O (40 mL) was added and the product was

extracted with CHCl₃, washed with H₂O, dried over

MgSO₄, and concentrated under reduced pressure. Ethyl

ether was added to the residue and evaporated to give the

1

isothiocyanate (0.47 g, 84%) as a white glassy solid; H

NMR (300 MHz, CDCl₃) d (mixture of conformers of

3:2 ratio) 7.62 and 7.51 (s, 1H, H-2⁰), 7.03–7.20 (m, 2H,

H-5⁰and H-6⁰), 6.62 and 6.60 (s, 1H, H-5), 6.41 and 6.29

(s, 1H, H-8), 5.21 and 5.07 (m, 1H, H-1), 4.2–2.1 (m,

6H, H-3, H-4 and CH₂Ar), 3.87, 3.81 and 3.72 (s, 6H,

MeO), 1.58, 1.43 and 1.30 (s, 9H, t-Bu); IR (KBr) 2082

(NCS), 1686 (C@O), 1518 (C@C Ar) cm^ꢀ1. Anal.

(C₂₄H₂₇IN₂O₄SÆ0.1CHCl₃) C, H, N.

5.1.15. 2-(9-Fluorenylmethoxycarbonyl)-1-(4-nitrobenzyl)-

1,2,3,4-tetrahydroisoquinoline-6,7-diol (20). 1 M solution

ofBBr₃inCH₂Cl₂(28 mL, 28 mmol)wasaddedtoacooled

solution (ꢀ78 ꢁC) of compound 19 (5.51 g, 9.36 mmol) in

100 mL of anhydrous CH₂Cl₂under an argon atmosphere.

The reaction mixture was stirred overnight at room tem-

perature followed by 40 mL of MeOH added upon cooling

(ꢀ78 ꢁC)andstirredatroomtemperaturefor5 h.Thesolu-

tion was then ﬁltered and evaporated four times with

MeOH. The product was recrystallized from MeOH–ethyl

ether mixture, to give 4.65 g (95%) of yellow crystals: mp

5.1.13.

1-(3-Iodo-4-isothiocyanatobenzyl)-6,7-dihydroxy-

1,2,3,4-tetrahydroisoquinoline hydrobromide (18). To a cold

solution (ꢀ78 ꢁC) of the isothiocyanate 17 (0.52 g,

0.92 mmol) in anhydrous CH₂Cl₂(15 mL) was added 1 M

solution of BBr₃in CH₂Cl₂(2.8 mL, 2.8 mmol) under argon

atmosphere. The mixture was stirred overnight at room tem-

perature followed by cooling it again (ꢀ78 ꢁC) and dry

MeOH (10 mL) was added. The mixture was stirred at room

temperature for 1 h. Solvents were evaporated under re-

duced pressure at 25 ꢁC and MeOH was added again and

evaporated. This procedure was repeated three times. Ethyl

ether was added to the oily residue. The oﬀ-white crystals

formed were ﬁltered and washed with ethyl ether yielding

1

216–218 ꢁC; H NMR (300 MHz, DMSO-d₆) d (mixture

of conformers of 1:1 ratio) 8.87 (br s, OH) 8.67 (br s,

OH), 8.04 (d, J = 8.7 Hz, ArH), 7.87 (d, J = 7.5 Hz,

ArH), 7.82 (d, J = 7.5 Hz, ArH), 7.52 (m, ArH), 7.12–

7.43 (m, ArH), 6.54 (s, ArH), 6.46 (s, ArH), 6.35 (s,

ArH), 5.15 (m, H-1), 4.76 (m, H-1), 4.02–4.34 (m, CH),

3.58–3.82 (m, CH), 2.83–3.40 (m, CH), 2.32–2.60 (m,

CH); ¹³C NMR (75 MHz, DMSO-d₆) d 154.47 and

154.38 (C@O), 146.87, 146.69, 145.99, 144.22, 144.05,

143.85, 143.75, 143.50, 143.40, 140.74, 140.70, 130.58,

127.55, 127.01, 126.60, 126.23, 124.86, 124.74, 124.54,

124.29, 124.24, 122.93, 120.08, 120.04, 115.19, 113.93,

66.38 and 66.06 (OCH₂), 55.17 and 54.78 (C-1), 46.72

and 46.60 (CHAr₂), 41.50 and 41.35 (C-3), 37.91 and

37.72 (CH₂Ar), 26.94 and 26.56 (C-4); IR (KBr) 3395

1

0.47 g (84%) of the title compound: mp 246–247 ꢁC; H

NMR (300 MHz, DMSO-d₆) d 9.12 (br s, 1H, OH), 8.85

+

(br s, 2H, NH₂, 8.57 (br s, 1H, OH), 7.96 (d, J_m= 1.7 Hz,

1H, H-2⁰), 7.52 (d, J_o= 8.2 Hz, 1H, H-5⁰), 7.42 (dd,

J_m= 1.7 Hz, J_o= 8.2 Hz, H-6⁰), 6.56 (s, 1H, H-5), 6.52 (s,

1H, H-8), 4.62 (br m, 1H, H-1), 3.5–2.7 (m, 6H, 3· CH₂);

¹³C NMR (75 MHz, DMSO-d₆) d 145.18, 144.05, 140.21,

137.77,134.11, 132.31,130.95, 127.04,122.31, 122.27,

115.18, 113.48, 96.44, 54.80, 39.00, 38.29, 24.19; IR (KBr)

+

3444 (OH), 3165, 2047 (NCS), 1589 (NH₂bend), 1522

(C@C Ar) cm^ꢀ1. Anal. (C₁₇H₁₅IN₂O₂SÆHBr) C, H, N.

and 3204 (OH), 1649 (C@O), 1519 and 1346 (NO₂) cm^ꢀ1

.

Anal. (C₃₁H₂₆N₂O₆) C, H, N.

5.1.14. 2-(9-Fluorenylmethoxycarbonyl)-6,7-dimethoxy-1-

(4-nitrobenzyl)-1,2,3,4-tetrahydroisoquinoline (19). A solu-

tion of 7 (3.28 g, 10 mmol) in 50 mL of CH₂Cl₂was added

dropwise to a cold (0 ꢁC) solution of Fmoc-Cl (2.72 g,

10.5 mmol) in 50 mL of CH₂Cl₂. The mixture was stirred

for 5 min followed by the addition of a solution of DIPEA

5.1.16. 6,7-Bis(ethoxycarbonyloxy)-2-(9-ﬂuorenylmethoxy-

carbonyl)-1-(4-nitrobenzyl)-1,2,3,4-tetrahydroisoquinoline

(21). Pyridine (0.55 g, 7 mmol) was added to a cold (0 ꢁC)

solution of the catechol 20 (1.05 g, 2 mmol) and ethylchlo-

roformate (0.65 g, 6 mmol) in 20 mL of CH₂Cl₂. The mix-

1694

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

ture was stirred overnight at room temperature. The solu-

tion was washed twice with water, twice with 1 N HCl,

water (2·), dried over MgSO₄, ﬁltered, and evaporated.

Traces of solvent were removed on pump and drying unit

to give 1.26 g (95%) of a white glassy solid, which starts

melting at 58 ꢁC. Long standing in hexanes gave crystals:

mp 134–136 ꢁC; ¹H NMR (300 MHz, CDCl₃) d (mixture

of conformers of 5:6 ratio) 8.07 (d, J = 8.4 Hz, ArH), 7.95

(d, J = 8.4 Hz, ArH), 7.73 (t, J = 7.3 Hz, ArH), 7.16–7.57

(m, ArH), 7.05 (s, ArH), 6.99 (s, ArH), 6.77 (s, ArH), 6.76

(s, ArH), 6.73 (s, ArH), 6.55 (s, ArH), 5.37 (t, J = 6.4 Hz,

H-1), 4.88 (dd, J = 11.0, 4.1 Hz, H-1), 4.17–4.54 (m,

CH₂O+CH), 4.07 (m, CH), 3.93 (m, CH), 3.75 (m, CH),

3.07–3.33 (m, CH), 2.93–3.03 (m, CH), 2.65–2.83 (m,

CH), 2.43–2.64 (m, CH), 1.40 (m, Me); ¹³C NMR

(75 MHz, CDCl₃) d 155.20 and 154.91 (NCO₂), 152.65

(OCO₂), 146.85, 146.62, 145.12, 145.00, 144.15, 143.81,

143.75, 143.02, 141.65, 141.33, 141.27, 141.13, 141.07,

140.50, 140.32, 134.13, 133.75, 133.23, 133.01, 130.44,

130.17, 127.73, 127.63, 127.13, 127.03, 126.96, 124.76,

124.46, 124.35, 123.51, 123.33, 123.17, 123.00, 121.71,

121.24, 120.04, 119.96, 67.28 and 66.05 (NCO₂CH₂),

65.32 (CH₂OCO₂), 55.60 and 55.37 (C-1), 47.92 and

47.25 (CHAr₂), 42.29 and 42.03 (C-3), 38.76 and 37.60

(CH₂Ar), 27.94 and 27.49 (C-4), 14.13 (Me); IR (KBr)

1772 (OCO₂), 1701 (NCO₂), 1520 and 1346 (NO₂), 1257

(C–O) cm^ꢀ1. Anal. (C₃₇H₃₄N₂O₁₀) C, H, N.

3371 (NH₂), 1770 (OCO₂), 1696 (NC@O), 1618, 1499,

1259 (C–O) cm^ꢀ1. Anal. (C₃₇H₃₅IN₂O₈) C, H, N.

5.1.18. 1-(4-Azido-3-iodobenzyl)-6,7-bis(ethoxycarbonyl-

oxy)-2-(9-ﬂuorenylmethoxycarbonyl)-1,2,3,4-tetrahydro-

isoquinoline (23). Isoquinoline 22 (0.153 g, 0.2 mmol) was

dissolved in 4 mL of AcOH, 1 mL of water was added and

stirred on an ice bath. A solution of NaNO₂(0.028 g,

0.4 mmol) was added and stirred for 15 min, followed

by 12 mL of cold water (0 ꢁC) was added. A solution of

NaN₃(0.104 g, 1.6 mmol) in 1 mL of water was added

and stirred for 2 h. The white precipitate was ﬁltered,

washed with water (4·), and dried in vacuum. Yield

0.154 g (97%): mp 61–63 ꢁC; ¹H NMR (300 MHz,

CDCl₃) d (mixture of conformers of 10:13 ratio) 7.70–

7.80 (m, ArH), 7.20–7.60 (m, ArH), 6.93–7.10 (m, ArH),

6.81–6.90 (m, ArH), 6.68–6.77 (m, ArH), 5.32 (t,

J = 6.5 Hz, H-1), 4.77 (dd, J = 10.8, 4.9 Hz, H-1), 4.62

(m, CH), 4.20–4.50 (m, CH₂O+CH), 3.94–4.13 (m, CH),

3.84 (m, CH), 3.32 (m, CH), 3.00 (m, CH), 2.44–2.82

(m, CH), 1.40 (m, Me); ¹³C NMR (75 MHz, CDCl₃) d

155.20 and 155.01 (NCO₂), 152.71 (OCO₂), 144.23,

144.02, 143.78, 143.08, 141.52, 141.32, 141.05, 140.99,

140.75, 140.48, 140.38, 140.09, 140.00, 135.92, 135.87,

134.56, 134.22, 133.28, 132.98, 130.81, 130.61, 127.68,

127.66, 127.25, 127.08, 126.93, 124.94, 124.89, 124.66,

124.57, 123.20, 122.94, 121.77, 121.30, 120.07, 119.97,

118.19, 118.02, 87.44 and 87.36 (C-3⁰), 67.44 and 66.49

(NCO₂CH₂), 65.30 (CH₂OCO₂), 55.63 and 55.37 (C-1),

47.91 and 47.29 (CHAr₂), 41.27 and 41.03 (C-3), 38.63

and 37.30 (CH₂Ar), 28.04 and 27.58 (C-4), 14.16 (Me);

IR (KBr) 2121 (N₃), 1771 (OCO₂), 1699 (NC@O), 1258

(C–O) cm^ꢀ1. Anal. (C₃₇H₃₃IN₄O₈) C, H, N.

5.1.17. 1-(4-Amino-3-iodobenzyl)-6,7-bis(ethoxycarbonyl-

oxy)-2-(9-ﬂuorenylmethoxycarbonyl)-1,2,3,4-tetrahydro-

isoquinoline (22). (1) The solution of compound 21

(1.00 g, 1.3 mmol) in 50 mL of EtOAc was hydrogenat-

ed at 60 psi with 10% Pd/C (0.35 g) for 4 h. The reaction

mixture was ﬁltered through Celite and evaporated in

vacuum. A glossy solid was stirred overnight with hex-

anes to give a white crystalline solid, yield 0.95 g

(99%), mp 127–129 ꢁC. The compound was used with-

out further puriﬁcation. (2) The solution of this aniline

(0.83 g, 1.3 mmol), BTMAICl₂(0.50 g, 1.43 mmol),

and CaCO₃(0.19 g, 1.95 mmol) in CH₂Cl₂(30 mL)

and MeOH (12 mL) was stirred for 2 h at room temper-

ature, ﬁltered, washed with satd Na₂SO₃(2·), water

(2·), dried over MgSO₄, ﬁltered, concentrated, and puri-

ﬁed by ﬂash chromatography on silica gel (EtOAc/hex-

anes,1:2) to aﬀord after stirring in hexanes a white

glassy solid (0.64 g, 64%): mp 73–75 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d (mixture of conformers of 5:7 ra-

tio) 7.70–7.79 (m, ArH), 7.50–7.59 (m, ArH), 7.20–

7.46 (m, ArH), 7.03 (d, J = 9.4), 6.78–6.86 (m, ArH),

6.53–6.63 (m, ArH), 5.30 (t, J = 6.6 Hz, H-1), 4.80 (m,

H-1), 4.46–4.60 (m, CH), 4.19–4.40 (m, CH₂O+CH),

3.81–4.13 (m, CH), 3.32 (m, CH), 3.06 (m, CH), 2.93

(d, J = 6.6 Hz, CH₂Ar), 2.47–2.80 (m, CH), 1.40 (m,

Me); ¹³C NMR (75 MHz, CDCl₃) d 155.19 (NCO₂),

152.74 (OCO₂), 145.33, 144.39, 144.19, 143.84, 143.27,

141.40, 141.33,140.86, 140.34, 139.62, 139.40, 135.09,

134.82, 133.32, 133.04, 130.60, 130.47, 129.08, 127.58,

127.18, 127.08, 126.92, 125.06, 124.96, 124.82, 124.77,

123.12, 122.78, 121.80, 121.40, 120.00, 119.92, 114.60,

114.44, 83.93 and 83.84 (C-3⁰), 67.37 and 66.85

(NCO₂CH₂), 65.26 (CH₂OCO₂), 55.80 (C-1), 47.74 and

47.33 (CHAr₂), 40.96 (C-3), 38.61 and 37.23 (CH₂Ar),

28.08 and 27.75 (C-4), 14.16 (Me); IR (KBr) 3457 and

5.1.19. 1-(4-Azido-3-iodobenzyl)-1,2,3,4-tetrahydroisoquin-

oline-6,7-diol hydrochloride (24). Pyrrolidine (3 mL) was

added to a solution of isoquinoline 23 (0.279 g, 0.35 mmol)

in 3 mL of CH₂Cl₂under an argon atmosphere and stirred

for1 hatroomtemperature. Thereactionmixturewascon-

centrated and then dried on a vacuum pump. The resulting

oil was puriﬁed by ﬂash chromatography on silica gel

(EtOAc/MeOH, 20:1, 10:1). Homogeneous fractions were

evaporated, dissolved in MeOH, and 1 M HCl in ethyl

ether (0.5 mL, 0.5 mmol) was added. More ether was add-

ed to initiate crystallization. Yield 0.072 g (44%): mp 188–

1

190 ꢁC (dec.); H NMR (300 MHz, DMSO-d₆) d 9.14 (s,

1H, OH), 8.93 (br s, 1H, NH), 8.88 (s, 1H, OH), 8.74 (br

s, 1H, NH), 7.87 (d, J = 1.8 Hz, 1H, ArH), 7.44 (dd,

J = 8.2, 1.8 Hz, 1H, ArH), 7.35 (d, J = 8.2 Hz, 1H, ArH),

6.56 (s, 1H, ArH), 6.54 (s, 1H, ArH), 4.57 (m, 1H, CH),

2.30–3.30 (m, 6H, CH); ¹³C NMR (75 MHz, methanol-

d₄) d 147.04 and 145.80 (C-6 and C-7), 142.81 (C-1⁰),

142.18 (C-2⁰), 135.24 (C-4⁰), 132.16 (C-6⁰), 123.68

and 123.19 (C-4a and C-8a), 120.10 (C-5⁰), 116.27 (C-5),

114.25 (C-8), 89.00 (C-3⁰), 57.48 (C-1), 40.88 (C-3), 39.92

(CH₂Ar), 25.63 (C-4); IR (KBr) 3600–2300 (br, OH,

NH), 2122 (N₃), 1611, 1528, 1483 cm^ꢀ1. Anal. (C₁₆H₁₅I-

N₄O₂ÆHClÆ0.5 Et₂O) C, H, N.

5.1.20. N-(3,4-Dimethoxyphenethyl)-4-benzoylphenylace-

tamide (26). A solution of p-benzoylphenylacetic acid²⁶

(4.81 g, 0.02 mol) and SOCl₂(4.02 g, 0.034 mol) in ben-

zene (40 mL) was heated at reﬂux for 3 h, evaporated

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1695

two times with benzene, dissolved in CH₂Cl₂(50 mL),

cooled on an ice bath, and 3,4-dimethoxyphenetylamine

(3.99 g, 0.022 mol) was added dropwise. Et₃N (4.05 g,

0.04 mol) was added and the reaction mixture was stirred

overnight at room temperature, washed with 1 N HCl

(2·), H₂O, 1 N NaOH, and H₂O, dried over MgSO₄,

and evaporated. The resulting oil was recrystallized from

CH₂Cl₂–hexanes mixture. Yield 6.72 g (83%): mp 113–

MgSO₄, ﬁltered, and concentrated. The resulting oil

was dissolved in CHCl₃(75 mL) and a solution of

Hg(ClO₄)₂ÆxH₂O (9.61 g, about 21 mmol) in MeOH

(75 mL) was added and stirred for 1 h. A yellow pre-

cipitate was removed by ﬁltering through Celite. The

ﬁltrate was extracted with CHCl₃, satd Na₂CO₃, brine,

dried over MgSO₄, ﬁltered, and concentrated to an ap-

prox. 10 mL volume. A solution of oxalic acid (1.26 g,

10 mmol) in MeOH (10 mL) was added followed by

some ethyl ether. After cooling, the white crystals were

ﬁltered and washed with ethyl ether. Yield 2.35 g

1

114 ꢁC; H NMR (300 MHz, CDCl₃) d 7.72–7.81 (m,

4H, ArH), 7.61 (m, 1H, ArH), 7.46–7.53 (m, 2H, ArH),

7.32 (d, J = 8.3 Hz, 2H, ArH), 6.73 (d, J = 8.1 Hz, 1H,

ArH), 6.65 (d, J = 1.9 Hz, 1H, ArH), 6.56 (dd, J = 8.1,

1.9 Hz, 1H, ArH), 5.48 (br t, 1H, NH), 3.83 (s, 3H,

MeO), 3.82 (s, 3H, MeO), 3.60 (s, 2H, CH₂CO), 3.48

(m, 2H, CH₂N), 2.71 (t, J = 6.9 Hz, 2H, CH₂Ar); ¹³C

NMR (75 MHz, CDCl₃) d 196.13 (C@O), 169.85

(NCO), 149.08 (O–C, Ar), 147.75 (O–C, Ar), 139.53,

137.40, 136.55, 132.51, 130.93, 130.63, 129.94, 129.27,

128.32, 120.58, 111.75, 111.28, 55.87 (MeO), 55.83

(MeO), 43.73, 40.80, 35.02 (CH₂Ar); IR (KBr) 3327

(NH), 1659 (C@O), 1642 (C@O, amide), 1549,

1518 cm^ꢀ1. Anal. (C₂₅H₂₅NO₄Æ0.05 CH₂Cl₂) C, H, N.

1

(72%): mp 201–202 ꢁC; H NMR (300 MHz, DMSO-

d₆) d 8.24 (br s, NH), 7.65–77.6 (m, 5H, ArH), 7.49–

7.61 (m, 4H, ArH), 6.79 (s, 1H, ArH), 6.49 (s, 1H,

ArH), 4.73 (t, J = 7.0 Hz, 1H, H-1), 3.73 (s, 3H,

MeO), 3.54 (s, 3H, MeO), 3.38–3.49 (m, 2H, CH),

3.19–3.32 (m, 2H, CH), 2.82–3.01 (m, 2H, CH); ¹³C

NMR (75 MHz, DMSO-d₆) d 195.48 (C@O), 164.39

(HO₂CCO₂H), 148.20 and 146.99 (C-6 and C-7),

141.64, 137.05, 135.77, 132.68, 130.01, 129.89,

129.51,128.57, 124.19 and 124.01 (C-4a and C-8a),

111.74 (C-5), 110.14 (C-8), 55.44 (OMe), 55.27

(OMe), 54.77 (C-1), 38.38 (CH₂Ar), 24.67 (C-4); IR

(KBr) 3300–2300 (NH, OH), 1721 (C@O, acid), 1657

(C@O), 1609, 1521 cm^ꢀ1. Anal. (C₂₅H₂₅NO₃Æ(COOH)₂)

C, H, N.

5.1.21.

6,7-Dimethoxy-1-[4-(2-phenyl-1,3-dithiolane-2-

yl)benzyl]-1,2,3,4-tetrahydroisoquinoline oxalate (27). A

solution of the amide 26 (4.84 g, 12 mmol), 1,2-ethanedith-

iol (8.0 g, 82 mmol), and BF₃ÆEt₂O (7.2 mL, 8.3 g,

59 mmol) in 18 mL of anhydrous CHCl₃was stirred over-

night at room temperature under an argon atmosphere.

CHCl₃was added, washed three times with satd Na₂CO₃,

brine, dried over MgSO₄, and concentrated. The resulting

oil was dissolved in 50 mL of MeCN and reconcentrated,

MeCN (85 mL) and POCl₃(7.8 mL) were added and re-

ﬂuxed for 5 h. The solution was concentrated and evapo-

rated with MeOH (50 mL) three times. The residue was

dissolved in MeOH (125 mL) and NaBH₄(9.6 g,

0.25 mol) was added in small portions. The reaction mix-

ture was stirred overnight at room temperature. MeOH

was evaporated, CHCl₃added, and washed twice with

10% NaOH, H₂O, dried over MgSO₄, ﬁltered, and concen-

trated. The product was dissolved in CH₂Cl₂and MeOH,

and solution of oxalic acid (1.8 g, 14 mmol) in minimal

amount of MeOH was added followed by ethyl ether.

The white crystals were ﬁltered and washed with ethyl

5.1.23. 4-(6,7-Dihydroxy-1,2,3,4-tetrahydroisoquinoline-

1-yl-methyl)benzophenone hydrobromide (29). The free

base was obtained by extraction of a solution of isoquin-

oline salt 28 (0.478 g, 1 mmol) in 1 N NaOH with CHCl₃,

dried over MgSO₄, evaporated, and dried in vacuum. The

resulting oil was dissolved in anhydrous CH₂Cl₂(20 mL)

under an argon atmosphere and cooled down with dry ice

bath. Solution of 1 N BBr₃in CH₂Cl₂(4.0 mL, 4 mmol)

was added with syringe and stirred overnight at room

temperature. Cooled down again followed by MeOH

(10 mL) was added and stirred for 4 h at room tempera-

ture, and evaporated ﬁve times with MeOH almost to dry-

ness. MeOH–ethyl ether mixture was added to crystals.

After refrigeration, crystals were collected by ﬁltration

and washed with MeOH–ethyl ether. The product was

recrystallized from MeOH–ether. Yield 0.388 g (88%):

mp 239–241 ꢁC; ¹H NMR (300 MHz, methanol-d₄) d

7.79 (m, 4H, H-3⁰+H-5⁰+H-2⁰⁰+H-6⁰⁰), 7.65 (m, 1H, H-

4⁰⁰), 7.48-7.58 (m, 4H, H-2⁰+H-6⁰+H-3⁰⁰+H-5⁰⁰), 6.64 (s,

1H, ArH), 6.52 (s, 1H, ArH), 7.65 (m, 1H, H-1), 3.50–

3.60 (m, 2H, H-3), 3.19–3.37 (m, 2H, CH), 2.88–2.32

(m, 2H, CH); ¹³C NMR (75 MHz, methanol-d₄) d

198.16 (C@O), 147.05 and 145.76 (C-6 and C-7), 141.95,

138.74, 138.19, 133.93 (C-4⁰⁰), 131.77, 131.02, 130.89,

129.57, 123.71 and 123.30 (C-4a and C-8a), 116.30 (C-

5), 114.35 (C-8), 55.45 (C-1), 41.11 (C-3), 40.87 (CH₂Ar),

25.63 (C-4); IR (KBr) 3600–2600 (OH+NH), 1646

(C@O), 1608 (C@C, Ar), 1527, 1282 cm^ꢀ1. Anal.

(C₂₃H₂₁NO₃ÆHBr) C, H, N.

1

ether. Yield 3.93 g (59%): mp 151–152.5 ꢁC; H NMR

(300 MHz, DMSO-d₆) d 7.43–7.54 (m, 4H, ArH), 7.20–

7.36 (m, 5H, ArH), 6.77 (s, 1H, ArH), 6.17 (s, 1H, ArH),

4.63 (m, 1H, H-1), 3.71 (s, 3H, MeO), 3.39–3.50 (m, 1H,

CH), 3.40 (s, 4H, SCH₂), 3.39 (s, 3H, MeO), 3.12–3.31

(m, 3H, CH), 2.85–2.98 (m, 2H, CH); ¹³C NMR

(75 MHz, methanol-d₄) d 166.63 (HO₂CCO₂H), 150.51

(O–C, Ar), 149.00 (O–C, Ar), 146.23, 145.78, 136.11,

130.49, 129.91, 129.27, 128.93, 128.27, 124.93 and 124.56

(C-4a and C-8a), 112.98 and 111.54 (C-5 and C-8), 57.20

(C-1), 56.42 (MeO), 41.08 (SCH₂), 41.04 (SCH₂), 40.77

(C-3), 39.91 (CH₂Ar), 25.78 (C-4); IR (KBr) 3300–2300

(br, NH+OH), 1721 (C@O, acid), 1614, 1520 cm^ꢀ1. Anal.

(C₂₇H₂₉NO₂S₂Æ(COOH)₂) C, H, N.

5.2. Pharmacological studies

5.1.22. 4-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-

1-yl-methyl)benzophenone oxalate (28). Oxalic acid salt

of 27 (3.81 g, 6.88 mmol) was mixed with 1 N NaOH,

extracted with CHCl₃, washed with brine, dried over

5.2.1. Radioligand binding studies. The procedure for

handling of CHO cells expressing b-AR subtypes and

incubation with radioligand are as described previous-

ly.²⁸Brieﬂy, following growth of cells to 70% conﬂuen-

1696

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

cy, CHO cells expressing human b₁-, b₂-, or b₃-AR were

trypsinized and harvested into Ham’s F-12 solutions.

Cells were pelleted and washed three times with Tris–

EDTA buﬀer (pH 7.4; TRIZMA HCl, 50 mM; NaCl,

150 mM; disodium EDTAÆ2H₂O, 20 mM). Cells were

then suspended in Tris–EDTA buﬀer after centrifuga-

tion. Competition binding assays were performed at

37 ꢁC by incubating cells with varying concentrations

of drugs in the presence of [¹²⁵I]ICYP ((1.5–5 · 10⁴cells

/18–70 pM for human b₁-, or b₂-AR, (3–5) · 10⁵cells/

200–500 pM for human b₃-AR). Non-speciﬁc binding

was determined in the presence of (ꢀ)-propranolol

(1 lM for human b₁-, or b₂-AR, 100 lM for human

b₃-AR). The incubations were terminated by rapid ﬁltra-

tion over Whatman GF/C (for human b₃-AR, presoa-

ked in 0.1% polyethylenimine) glass ﬁber ﬁlters using a

Brandel model 12-R cell harvester. The ﬁlters were

washed three times with the Tris–EDTA buﬀer (4 ꢁC)

and dried under cell harvester vacuum. The radioactivity

in the ﬁlters was measured by gamma scintillation

counting using Beckmann gamma counter model 8000.

K_ivalues were calculated from the obtained IC₅₀values

by the method of Cheng and Prusoﬀ.²⁹

5.2.4. Time and concentration dependence of aﬃnity

binding. CHO cells expressing the human b₂-AR (about

30–40 · 10³cells/150 lL) were suspended in 1.2 mL Tris

buﬀer in microfuge tubes and incubated at room tem-

perature in a rotating shaker (Robbins Scientiﬁc) with

the acetamide 30 (K_i= 6.0 nM), chloroacetamide 6

(K_i= 4.3 nM) or isothiocyanate 18 (K_i= 45 nM) at con-

centrations of 3-, 10-, 30-, and 100-fold of the K_ivalues

for time periods ranging from 2 to 45 min. Incubations

were stopped by centrifugation of the cell suspension

at 1500g in microcentrifuge (Eppendorf Model 5415C).

Cell pellets were resuspended in 1.2 mL of fresh buﬀer

and the samples were placed again on the shaker for

about 15 min to allow for drug equilibrium (bound

and free drug), followed by recentrifugation and resus-

pension. This washing procedure was repeated three

times for each sample. Protein contents of the ﬁnal

reconstituted cell suspensions were determined by the

method of Lowry et al.³¹

Triplicate aliquots of normalized suspensions (20,000–

30,000 cells/aliquot) were incubated with 60–240 pM of

[

¹²⁵I]ICYP in a ﬁnal volume of 250 lL in buﬀer for 1 h

at 37 ꢁC. Binding reactions were terminated by rapid ﬁl-

tration (5 mL of ice-cold Tris buﬀer · 2 times) of the

samples through Whatman GF/B ﬁlters on a Brandel

Model 12-R cell harvester and the radioactivity present

on ﬁlters was measured in a gamma counter (Model

1470 Wizard, Wallac Inc., Gaithersburg, MD). Non-

speciﬁc binding for each sample aliquot was determined

in the presence of 2 · 10^ꢀ6M ( ) propranolol.

5.2.2. cAMP-RIA assay. The procedures for the han-

dling, incubation ,and assay of cAMP in CHO cells

expressing human b₁-, b₂-, or b₃-AR subtypes are as pre-

viously described.³⁰Cells were grown to conﬂuence in 60-

mm dishes, washed with Hanks’ balanced salt solution,

and then incubated with Hanks’ balanced salt solution

(pH 7.4) containing 20 mM HEPES, 1 mM 3-isobutyl-

1-methylxanthine (IBMX), and 1 mM ^L-ascorbic acid

for 30 min at 37 ꢁC. Varying concentrations (10^ꢀ11

–

5.2.5. Time- and concentration-dependent photoaﬃnity

binding. CHO Cells expressing the human b₂-AR (about

30–40 · 10³cells/150 lL) were suspended in 1.2 mL Tris

buﬀer in microfuge tubes and incubated for 1 h at room

temperature under red light in a rotating shaker (Rob-

bins Scientiﬁc, 18 rpm) with the azide 24 (K_i= 68 nM)

at concentrations of 1-, 3-, 10- and 100-fold of the K_i

value. The suspensions were then transferred to quartz

tubes and exposed to UV light at 350 nm for 30 s or

5 min. The photolyzed suspensions were then centri-

fuged for 2 min at about 1500g in microcentrifuge

(Eppendorf Model 5415C). Cell pellets were resuspend-

ed in 1.2 mL of fresh buﬀer and the samples were placed

again on the shaker for about 15 min in the dark to al-

low equilibrium of the drug between bound and free

forms, which was followed by recentrifugation and

resuspension. This washing procedure was repeated

twice for a total of three times for each sample. Protein

contents of the ﬁnal reconstituted cell suspensions were

determined by the method of Lowry et al.³¹

10^ꢀ4M) of the compounds were added with incubation

of an additional 30 min. After removal of the Hank’s buﬀ-

er, the cAMP generated within the cells was extracted by

the addition of trichloroacetic acid (6% w/v). The precip-

itated protein was dissolved in 0.1 N NaOH. Protein con-

tent was determined by the method of Lowry et al.³¹using

bovine serum albumin as a standard. cAMP levels in

CHO cells were determined using the radioimmunoassay

technique of Brooker et al.³²The amount of cAMP was

measured as the amount of ¹²⁵I-labeled succinyl-cAMP

tyrosine methyl ester/antibody precipitated.

5.2.3. CRE-LUC assay. CHO cells stably expressing

human b₁-, b₂-, or b₃-AR populations were transfec-

ted with a 6 CRE-LUC plasmid (gift from Dr.

Himmler A. Vienna, Austria) using electroporation

with a single 70 ms, 150 V pulse.³³The transfected

CHO cells were seeded at a density of 40,000/well

in 96-well microtiter plates (culturplate, Packard)

and allowed to grow for 20 h. After 20 h, the cells

were treated with varying drug concentrations

(10^ꢀ11–10^ꢀ4M) for 4 h. Following drug exposures,

the cells were lysed and luciferase activity was mea-

sured using the LucLite^ꢂassay kit (Packard). Chang-

es in light production were measured by a Topcount^ꢂ

luminometer (Packard). Data were analyzed in dupli-

cate at each concentration and expressed as a percent

luciferase response relative to the maximum response

to (ꢀ)-isoproterenol (10^ꢀ6M). Results are expressed

as means SEM of n = 5–16.

Triplicate aliquots of normalized suspensions (20,000–

30,000 cells/aliquot) were incubated with 60–240 pM

[

¹²⁵I]ICYP in a ﬁnal volume of 250 lL in buﬀer for

1 h at 37 ꢁC. Binding reactions were terminated and

radioactivity was measured as described earlier in

Section 5.2. Non-speciﬁc binding for each sample ali-

quot was determined in the presence of 2 · 10^ꢀ6M

( ) propranolol. In control experiments, untreated cell

suspensions were subjected to identical photolysis

procedure and evaluated for radioligand binding.

V. I. Nikulin et al. / Bioorg. Med. Chem. 14 (2006) 1684–1697

1697

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Acknowledgments

We are grateful for the support of this research from

the National Institute of Health (USPHS Grant HL-

22533), the Department of Pharmacology, and the

USDA ARS Agreement No. 58-6408-2-0009 in the

National Center for Natural Products Research at

the University of Mississippi. We also thank Dr. Rich-

ard Fertel (Department of Pharmacology, College of

Medicine, The Ohio State University, Columbus, OH)

for providing radiolabel and antibody for the cAMP-

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Article Doi

1-Benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diols as novel affinity and photoaffinity probes for β-adrenoceptor subtypes

DOI: 10.1016/j.bmc.2005.12.027

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Full text of DOI:10.1016/j.bmc.2005.12.027

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