Spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes) as inhibitors of gastric acid secretion

Bioorganic & Medicinal Chemistry 17 (2009) 368–384

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Bioorganic & Medicinal Chemistry

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Spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes) as inhibitors

of gastric acid secretion ^q

*

Andreas Marc Palmer , Sandra Chrismann, Gabriela Münch, Christof Brehm, Peter Jan Zimmermann,

Wilm Buhr, Jörg Senn-Bilﬁnger, Martin Philipp Feth, Wolfgang Alexander Simon

NYCOMED GmbH, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 2 June 2008

Accepted 23 October 2008

Available online 26 October 2008

Asymmetric and symmetric spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes) were prepared using a

synthetic approach that comprised a cross-metathesis reaction and an acid-catalyzed cycloisomerisation

as key steps. The target compounds constitute potent inhibitors of the gastric proton pump enzyme with

inhibitory activity comparable to potassium-competitive acid blockers (P-CABs) belonging to the known

9-aryl-7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridine series. Spiro(imidazo[1,2-a]pyrano[2,3-c]pyr-

idine-9,2⁰-indenes) represent the ﬁrst example for P-CABs, in which the distance between the heterocy-

clic scaffold and the aryl residue has been modiﬁed, and are promising candidates for further

development as anti-ulcer drugs.

Keywords:

Cross-metathesis

7H-8,9-Dihydropyrano[2,3-c]imidazo[1,2-

a]pyridines

Anti-secretory activity

Gastric acid pump

H⁺/K⁺-ATPase

1. Introduction

ment of SCH 28080 (1) was stopped due to extensive metabolism

and associated liver toxicity.¹⁴

Acid related diseases, such as gastroesophageal reﬂux disease

(GERD), have a high prevalence and inﬂuence strongly the quality

of life of the affected patients.^6–8The inhibition of the gastric pro-

ton pump enzyme (H⁺/K⁺-ATPase) represents a major approach in

the development of drugs against these medical conditions. Many

pharmaceutical companies have spent considerable efforts in the

identiﬁcation of irreversible and reversible inhibitors of the

H⁺/K⁺-ATPase. Substances belonging to the class of irreversible

inhibitors are referred to as proton pump inhibitors (PPIs) and have

been available as therapeutics for a long time already. Due to their

new mode of action, reversible inhibitors of the gastric acid pump

enzyme (potassium-competitive acid blockers, P-CABs) might be

able to overcome some limitations encountered during the treat-

ment with PPIs.^9–12

In the past two decades, one important approach for the identi-

ﬁcation of potent P-CABs relied on the structural class of substi-

tuted imidazo[1,2-a]pyridines. The inhibitor SCH 28080 (1)

represents the clinical prototype of this series (Fig. 1). SCH 28080

(1) inhibits the gastric proton pump enzyme (H⁺/K⁺-ATPase) by a

kinetically competitive and reversible inhibition mechanism with

respect to the potassium ion and shows excellent anti-secretory

and cytoprotective properties.^13–15However, the clinical develop-

One approach to synthesize advanced chemical analogues of

SCH 28080 was based on results from molecular modelling, which

suggested that in the gas phase, SCH 28080 could adopt various

‘folded’ conformations close to the global minimum of energy.^15,16

On the other hand, single-crystal X-ray analysis revealed that solid

SCH 28080 existed in an ‘extended’ conformation.^13,16By synthesis

of simple analogues, which imitated these conformations, it was

shown that an ‘extended’ relationship between the phenyl group

and the heterocyclic nucleus was required for effective binding

to H⁺/K⁺-ATPase.¹⁵Subsequently, the tricyclic imidazo[1,2-a]pyri-

dine 2 was synthesized in which the pyrano ring was considered

to enforce this requisite ‘extended’ relationship and to mimic a

7-methyl substituent which would be effective in overcoming

the toxic properties of 1 while retaining its desirable anti-secretory

effects (Fig. 1).^15,17

In the course of our P-CAB lead optimization project we synthe-

sized 7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridines of the

general formula 3, which were devoid of the critical cyanomethyl

group (Fig. 1).⁵Several derivatives 3 with R³= CH₃were shown

to be potent inhibitors of the H⁺/K⁺-ATPase with pIC₅₀values com-

parable to that of lead compound 2 (pIC₅₀of 2: 7.0).^5,18The pK_ava-

lue and the metabolic stability of the P-CABs of the formula 3 were

controlled by the nature of the substituent R⁶. A variety of tricyclic

imidazopyridines 3 that contained a carboxamide residue R⁶

showed promising pharmacological activity and favourable pK_a

values in the range of 6.4–7.2 (pK_aof 2: 6.0).^5,18The pK_avalue of

q

Design of novel P-CABs, Part VI, see Refs. 1–5.

* Corresponding author. Tel.: +49 7531 844783; fax: +49 7531 8492087.

E-mail address: andreas.palmer@nycomed.com (A.M. Palmer).

doi:10.1016/j.bmc.2008.10.055

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

369

considered to be one of the most interesting representative of

the (9S)-series 4 and an efﬁcient asymmetric synthesis was devel-

oped for this target compound.^19,20

CN

N

7

N

9-Aryl-substituted

7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-

Ph

O

a]pyridines 2–7 represent rigid analogues of SCH 28080 (1). We

were interested whether further rigidisation would result in an in-

creased afﬁnity of the corresponding P-CAB to the gastric proton

pump enzyme (H⁺/K⁺-ATPase) and, consequently, aiming at the

preparation of spiro-substituted 7H-8,9-dihydropyrano[2,3-c]imi-

dazo[1,2-a]pyridines 8 and 9 (Fig. 1). Depending on the attachment

point of the indane moiety, the series are referred to as ‘asymmet-

ric series’ 8 and ‘symmetric series’ 9, respectively. Here, we wish to

report on the synthesis and the biological activity of these novel P-

Ph

SCH 28080 (1)

("extended")

(" folded")

R³

CN

R⁶

N

CABs²¹

.

N

O

2. Results and discussion

Ph

Ar

2

3

2.1. Synthesis of spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-

indenes)

R³

R⁶

The synthetic approach towards spiro-substituted 7H-8,9-dihy-

dropyrano[2,3-c]imidazo[1,2-a]pyridines 8 and 9 is depicted in

Scheme 1. Key intermediates 12 are obtained by cross-metathesis

reaction between 7-allylimidazo[1,2-a]pyridine building blocks

10, in which the phenolic hydroxy function is protected with a

suitable group R, and methyleneindane derivatives 11. Cycloiso-

merisation of these unsaturated alcohols 12 should then be feasi-

ble in the presence of Brönsted or Lewis acids affording the

respective target compounds 8 or 9. A similar approach has already

proven its value in the synthesis of 9-aryl-substituted 7H-8,9-dihy-

dropyrano[2,3-c]imidazo[1,2-a]pyridines 3⁵: the cross-metathesis

of 10 with styrene derivatives 13 was accomplished in the pres-

ence of second-generation Grubbs catalyst. Heating of the obtained

unsaturated alcohols 14 in 85% phosphoric acid afforded the target

compounds 3. A selection of catalysts that are commonly used for

cross-metathesis reactions is depicted in Figure 2.^22–29

N

O

Ar

4

5

6 (R³=Me, Ar=Ph

7 (R³=Me, Ar=Ph

R⁶=C(O)NMe₂)

R⁶

N

O

N

O

R

2.1.1. Asymmetric series

9

The cross-metathesis reaction between 7-allyl-substituted imi-

dazo[1,2-a]pyridines, for example, 28–29, and substituted 1-meth-

yleneindanes, for example, 23–25, constituted the key step in the

synthesis of asymmetric spiro-substituted 7H-8,9-dihydropyr-

ano[2,3-c]imidazo[1,2-a]pyridines 8. The 1-methyleneindanes

23–25 were prepared in good yield by Wittig reaction of the

respective indan-1-one precursor 20–22 with methyltriphenyl-

phosphonium bromide using potassium tert-butylate as base

(Scheme 2).^30,31

The 7-allyl-substituted imidazo[1,2-a]pyridine 27 was prepared

by O-allylation of the 8-hydroxy derivative 26 and subsequent Cla-

isen rearrangement as described previously (Scheme 3).⁵In order

to avoid formation of substrate–catalyst complexes between imi-

dazo[1,2-a]pyridine 27 and the respective Ruthenium complex

15–19, the phenolic hydroxy group of 27 was protected either by

transformation with pivaloyl chloride or by treatment with

8

Figure 1.

a potassium-competitive acid blocker is an important parameter

for two reasons: ﬁrst, it determines the concentration of the

protonated species, which is the active form for inhibition of H⁺/

K⁺-ATPase in the parietal cell.^12,18Second, the parietal cell is distin-

guished from other compartments by its low pH value of approxi-

mately 3. Hence, P-CABs with low pK_avalues are accumulated in a

selective manner and possible interactions with enzymes that are

expressed in other cells are prevented, which should translate in

an improved safety proﬁle. It was also demonstrated that the

(9S)-enantiomers 4 were responsible for the gastric acid secretion

inhibiting action, whereas the (9R)-enantiomers 5 were virtually

inactive (Fig. 1).⁵The imidazo[1,2-a]pyridine BYK 311319 (6) was

R⁶

N

3

8, 9

N

OR

Ar

OR

13

Ar

R

14

10

11

12

R

Scheme 1.

370

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

80 °C for 2 h in the presence of 8 mol% of catalyst 15, the desired

Mes

N

N Mes

Mes

N

Mes

Ph

cross-coupling product 30 was isolated in 25% yield. More encour-

aging results were obtained in the case of silyl ether 29: the cross-

metathesis reaction of 23 and 29 in the presence of 9 mol% of cat-

alyst 15 (dichloromethane, 40 °C, 18 h) afforded oleﬁn 31 in 72%

yield. The cleavage of the protecting group was accomplished un-

der standard conditions: the unsaturated alcohol 32 was obtained

in good yield either by saponiﬁcation of pivaloyl ester 30 with

lithium hydroxide or by treatment of silyl ether 31 with tetrabutyl-

ammonium ﬂuoride. Finally, target compound 33 was obtained in

70% yield by gentle heating of a solution of alcohol 32 in 85%

phosphoric acid. This method has already proven its value for the

cycloisomerisation of alcohols 14 into 9-aryl-7H-8,9-dihydropyr-

ano[2,3-c]imidazo[1,2-a]pyridines 3. However, target compound

33 was isolated containing traces of the known imidazo[1,2-a]pyr-

idine 34. Latter compound could be removed by preparative HPLC

and was presumably formed by transfer of the styrene ligand from

catalyst 15 to the respective substrate 28 or 29 and subsequent

cyclisation of the intermediate alcohol 14 (Scheme 1, R⁶= CONMe₂,

Ar = Ph). Resolution of racemate 33 into the enantiomers 35 and 36

was accomplished by HPLC using a CHIRALPAK AD-H column.

When maleic acid was added to a solution of the free base 33 in

acetone, the maleate salt 37 of target compound 33 was isolated

in 73% yield.

PCy₃

Ru

Cl

Ru

PCy₃

R²

PCy₃

O

R¹

15

16: R¹= R²= H

17: R¹= Ph, R²= H

18: R¹= H, R²= NO₂

19

Figure 2.

O

(i)

R1

R2

20 (R1 = R2 = H)

21 (R1 = F, R2 = H)

22 (R1 = H, R2 = Me)

23 (R1 = R2 = H)

24 (R1 = F, R2 = H)

25 (R1 = H, R2 = Me)

Scheme 2. Reagents and conditions. (i) [MePPh₃]⁺Br^ꢀ, KOtBu, Et₂O, 2–20 h, 23: 61%,

24: 85%, 25: 97%.

We were then interested in the question whether the cross-

metathesis/cyloisomerisation sequence was also feasible using

ethyl ester 40 as substrate (Scheme 4). This approach would allow

the convenient synthesis of a large number of 6-carboxamide-

chloro(dimethyl)thexylsilane yielding ester 28 and silyl ether 29,

respectively. For both derivatives 28 and 29, the cross-metathesis

reaction with 1-methyleneindane was investigated in the presence

of second-generation Grubbs catalyst 15. When a solution of piva-

loyl ester 28 and oleﬁn 23 in 1,2-dichloromethane was heated to

substituted

spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-ind-

enes) by saponiﬁcation of ester 44 and subsequent coupling of

carboxylic acid 45 with different amines. The O-protected

O

(i), (ii)

(v) or (vi)

(iii) or (iv)

N

OH

OR

OH

26

27

28 (R = Piv)

29 (R = TxDMS)

O

N

(vii) or (viii)

(ix)

O

OR

OH

O

34 (byproduct)

30 (R = Piv)

32

33

31 (R = TxDMS)

O

N

O

N

(x)

N

(xi)

N

OH

33

O

35

36

37

Scheme 3. Reagents and conditions: (i) allyl bromide, K₂CO₃, DMF, rt, 18.5 h, 67%; (ii) melting, 160 °C, 0.75 h, 76%; (iii) synthesis of 28: pivaloyl chloride, K₂CO₃, acetone, rt,

0.75 h, 71%; (iv) synthesis of 29: chloro(dimethyl)thexylsilane, imidazole, DMF, rt, 1 h, 93%; (v) synthesis of 30: oleﬁn 23, catalyst 15, 1,2-dichloroethane, 80 °C, 2 h, 25%; (vi)

synthesis of 31: oleﬁn 23, catalyst 15, CH₂Cl₂, 40 °C, 18 h, 72%; (vii) deprotection of 30: LiOH, 1,4-dioxane, 100 °C, 0.5 h, 66%; (viii) deprotection of 31: TBAF, THF, rt, 1 h, 71%

(mixture with [NBu₄]OH); (ix) H₃PO₄, 50 °C, 1 h, 70%; (x) CHIRALPAK AD-H, n-heptane/2-PrOH/Et₂NH = 80:20:0.1, 47/44% yield, 99.4/99.8% ee; (xi) maleic acid, acetone, 73%.

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

371

Table 1

O

Preparation of target compounds by amide coupling.

(i), (ii)

Si

N

EtO

Compound

(CO)NR⁰R⁰⁰

Yield (%)

70

N

O

N

HO

OH

38

OR

39 (R = H)

41

46

H

(iii)

O

40 (R = TxDMS)

N

H

47

48

49

50

51

52

53

68

O

EtO

N

O

48^a

74

(iv) or (v)

N

(vii)

OR

O

F

N

42 (R = TxDMS)

43 (R = H)

(vi)

F

49

N

O

R1

O

55^a

78^a

81

R'O

N

(ix)

R2

N

O

N

44 (R' = Et)

45 (R' = H)

46-55

O

(viii)

(see Table 1)

O

N

Scheme 4. Reagents and conditions: (i) allyl bromide, K₂CO₃, DMF, rt, 2.5 h, 47%;

(ii) melting, 160 °C, 1.5 h, 81%; (iii) variant 1: chloro(dimethyl)thexylsilane, NEt₃,

CH₂Cl₂, rt, 18 h, 80% (mixture with 32 wt% of 41); variant 2: chloro(dimethyl)thex-

ylsilane, NaH, CH₂Cl₂, rt, 2 h, 74% (mixture with 10 wt% of 41); (iv) oleﬁn 23,

catalyst 15, CH₂Cl₂, 40 °C, 18 h, 68% (mixture with 41); (v) catalyst 16, CH₂Cl₂, 40 °C,

1 day, 45% (mixture with 40 and 41); (vi) TBAF, THF, rt, 1 h, 52%; (vii) cyclisation of

43: H₃PO₄, 50 °C, 1 h, 80%; cyclisation of 42: H₃PO₄, 50 °C, 1.5 h, 91%; (viii) KOH,

EtOH, H₂O, 60 °C, 0.5 h, 98%; (ix) TBTU, DIPEA, DMF, 40 °C, 1 h, then amine, rt, 1–3 h,

44–81% (see Table 1).

O

N

H

54

44

O

55

47^a

N

a

Puriﬁcation by preparative HPLC to remove the respective by-product 3.

7-allylimidazo[1,2-a]pyridine 40 was synthesized in three steps

from building block 38 applying the known sequence of O-allyla-

tion, Claisen rearrangement, and treatment with chloro

(dimethyl)thexylsilane in the presence of triethylamine or sodium

hydride. In both cases, mixtures of silyl ether 40 with di-

methyl(1,1,2-trimethylpropyl)silanol (41) were obtained. Since

41 did not interfere with the cross-metathesis reaction and could

be removed at a later stage of the synthesis, the protection step

was not optimized further. Good conversion (>80%) was achieved

in the reaction of oleﬁns 23 and 40 in the presence of second-gen-

eration Grubbs catalyst 15 and the metathesis product 42 was iso-

lated in 68% yield. However, as described earlier for target

compound 33, product 42 contained traces of a by-product formed

by cross-metathesis reaction of the styrene ligand of catalyst 15

with substrate 40. This problem (tedious removal of the by-prod-

uct in all steps) was overcome by the use of Hoveyda–Grubbs cat-

alyst 16. Despite its lower activity (>50% conversion of 40), this

catalyst was used for future experiments. Heating a solution of ole-

ﬁns 23 and 40 and catalyst 16 in dichloromethane for 1 day at

40 °C afforded the cross-metathesis product 42 in 45% yield. The

use of more reactive metathesis catalysts was also investigated:

however, in the presence of 4 mol% of catalysts 17 or 18, the

cross-metathesis reaction between oleﬁns 23 and 40 (dichloro-

methane, 40 °C, 3 h) stalled at 40–50% conversion and similar con-

version rates were observed after a prolonged reaction time of

3 days. Imidazopyridine 44 was then secured by deprotection of si-

lyl ether 42 with tetrabutylammonium ﬂuoride and cyloisomerisa-

tion of the resulting unsaturated alcohol 43 in 85% phosphoric acid.

Spiro-imidazopyridine 44 could also be obtained by direct treat-

ment of silyl ether 42 with 85% phosphoric acid: in this case,

deprotection and cyclisation proceeded in one pot and the desired

product 44 was isolated in 91% yield. After saponiﬁcation of the

ester function, carboxamides 46–55 were obtained in good yields

by reaction of carboxylic acid 45 with the respective amine and

TBTU as coupling agent (Table 1).³²

In order to modify the substitution pattern of the indane moiety

of target compounds 8, the substituted 1-methyleneindanes 24

and 25 (Scheme 2) were used as coupling partners of 7-allyl-imi-

dazo[1,2-a]pyridine 40 (Scheme 5). Without optimization of the

reaction conditions (Hoveyda–Grubbs catalyst 16, dichlorometh-

ane, 40 °C, 17–24 h) the corresponding products 56 and 57 of the

cross-metathesis reaction were obtained in modest yield as mix-

tures with untransformed starting material 40. As already de-

scribed for the preparation of target compounds 33 and 44,

cycloisomerisation of the unsaturated alcohols 56 and 57 was like-

wise feasible in 85% phosphoric acid (50 °C, 2–2.5 h). Saponiﬁca-

tion of the ester moiety of the obtained spiro derivatives 58 and

59 was accomplished with potassium hydroxide. Finally, target

372

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

O

(i)

O

EtO

N

(i)

N

EtO

N

64

65

OTxDMS

N

OTxDMS

R2

40

(iii)

OR

R1

Br

56 (R1 = F, R2 =H)

57 (R1 = H, R2 = Me)

66 (R = H)

68 (R = THP)

69 (R = H)

(ii)

(iv)

67 (R = THP)

(ii)

(v)

O

(vi)

N

RO

N

Cl

N

(iv)

O

65

70

Scheme 6. Reagents and conditions: (i) TiCl₄, Mg, CH₂Cl₂, THF, 0 °C, 0.75 h, rt, 0.5 h,

15%; (ii) DHP, Amberlyst 15, n-hexane/Et₂O, rt, 45 min, 71%; (iii) Mg, THF, rﬂ., 1.5 h;

then allyl bromide, THF, rt, 17 h, 95%; (iv) HCl, MeOH, 50 °C, 20 h, 64%; (v) SOCl₂,

CH₂Cl₂, rt, 2 h, 89%; (vi) Pd(PPh₃)₄, NEt₃, CH₃CN, 85 °C, 70 min, 75%.

R1

R2

62 (R1 = F, R2 =H)

63 (R1 = H, R2 = Me)

58 (R = Et, R1 = F, R2 = H)

59 (R = Et, R1 = H, R2 = Me)

(iii)

Grubbs catalyst 15 and the desired heterodimer 71 was isolated

in 40–44% yield (1,2-dichloroethane, 80 °C, 2 h, see Scheme 7).

No signiﬁcant increase in conversion was observed when the ob-

tained mixture of oleﬁns 28 and 71 was puriﬁed and the metathe-

sis reaction repeated under identical conditions using this mixture

as starting material.

In order to investigate the amount of 2-methyleneindane (65)

that would be available in the course of the metathesis reaction,

the reaction rate of the competing homodimerization was judged

by heating 2-methyleneindane (65) in the presence of second-

generation Grubbs catalyst 15 (1,2-dichloroethane, 80 °C, 2 h).³⁵

The homodimer 72 was isolated in 78% yield (Scheme 7). No

product 71 was formed in the attempted cross-metathesis reac-

tion between 7-allyl-imidazo[1,2-a]pyridine 28 and homodimer

72.

60 (R = H, R1 = F, R2 = H)

61 (R = H, R1 = H, R2 = Me)

Scheme 5. Reagents and conditions: (i) preparation of 56: oleﬁn 24, catalyst 16,

CH₂Cl₂, 40 °C, 24 h, 34% (mixture with 40), preparation of 57: oleﬁn 25, catalyst 16,

CH₂Cl₂, 40 °C, 17 h, 43% (mixture with 40); (ii) H₃PO₄, 50 °C, 2–2.5 h, 58: further

transformation as crude product, 59: 38%; (iii) KOH, EtOH, H₂O, 60 °C, 1.5–2 h, 60:

28%, 61: 98%; (iv) TBTU, DIPEA, DMF, 40 °C, 1 h, then dimethylamine, rt, 1–3 h, 62:

38%, 63: 80%.

R

compounds 62 and 63, bearing a 5⁰-ﬂuoro-/a 4⁰-methyl-substitu-

ent, respectively, were obtained by TBTU-mediated coupling of

carboxylic acids 60 and 61 with dimethylamine.

2.1.2. Symmetric series

The synthesis of symmetric spiro-imidazo[1,2-a]pyridines 9

was performed in analogy to the method described for the prepa-

ration of asymmetric derivatives 8. However, both the cross-

metathesis and the cyclisation step turned out to be more

challenging.

Since the highest concentration of 2-methyleneindane (65) is

available in the beginning of the reaction we reasoned that the

use of more reactive metathesis catalysts should result in an in-

creased yield of 71. However, comparable conversion was observed

using catalyst 18 (0.5 g scale, 5.0 equiv of 65, 4ꢁ 1 mol% of catalyst

18, 1,2-dichloroethane, 80 °C, 2 h, 19% yield). In the presence of

10 mol% of Neolyst catalyst 19, no product 71 was formed under

otherwise identical reaction conditions.

In a multi-gram scale (8–10 g), two problems were encoun-

tered when the cross-metathesis reaction between 28 and 65

was performed in the presence of second-generation Grubbs cat-

alyst 15: ﬁrst, the yield of heterodimer 71 decreased to 20–24%.

Second, in analogy to the asymmetric series, mixtures of product

71 and by-product 14 (see Scheme 1: R⁶= CONMe₂, Ar = Ph),

resulting from ligand transfer from catalyst 15 to substrate 28,

were obtained, which were difﬁcult to separate. For this reason

Hoveyda–Grubbs catalyst 16 was used (addition in four portions).

A constant concentration of the cross-coupling partner in the

reaction mixture was ensured by continuous addition of 2-methy-

leneindane (65) over a period of 4 h. The oleﬁn 71 was isolated in

27% yield (8 g scale) and the pivaloyl protecting group removed

under basic conditions affording the unsaturated alcohol 73.

Two methods were investigated for the preparation of 2-methy-

leneindane (65), which was required as coupling partner in the

cross-metathesis reaction (Scheme 6). In our hands, the one-step

transformation of 1,3-dihydro-2H-inden-2-one (64) with magne-

sium and titanium tetrachloride proceeded in a less reliable man-

ner than described in the literature and 2-methyleneindane (65)

was obtained in 7–20% yield only.³³We therefore decided to pre-

pare reagent 65 following the alternative pathway depicted in

Scheme 6³⁴: ﬁrst aryl bromide 67, obtained by THP-protection of

2-bromobenzyl alcohol (66), was converted with magnesium and

allyl bromide affording Grignard product 68. After removal of the

THP protecting group, the resulting alcohol 69 was activated with

thionyl chloride and 2-methyleneindane (65) was secured by intra-

molecular Heck reaction of benzyl chloride 70.

In small scale (400–640 mg), the cross-metathesis reaction be-

tween 28 and 1.5–4 equiv of 2-methyleneindane (65) did not go

to completion in the presence of 8–10 mol% of second-generation

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

373

Table 2

O

Acid-catalyzed cycloisomerisation of the unsaturated alcohol 73.

(i) or (ii)

N

Entry

Conditions

Result

N

1

2

3

4

5

6

7

8

HCOOH, microwave, 150 °C, 0.5 h

H₂SO₄/HOAc = 1:2, 50 °C, 20 min

H₂SO₄/HOAc = 1:2, 80 °C, 10 min

H₃PO₄, 50 °C, 0.25 h

H₃PO₄, 80 °C, 0.5 h

H₃PO₄, 130 °C, 15 min

18% 75

10% 74

16% 74

15% 74

10% 74

17% 74

11% 74

8% 74

OPiv

28

71

HBr, [(n-C₁₆H₃₃)Bu₃P]⁺Br^ꢀ, 110 °C, 2 h

HSO₃F, nitropropane, ꢀ75 °C, 0.5 h

1.1 equiv Sn(OTf)₄, nitromethane, 120 °C, 0.75–1 h

20.0 equiv BF₃–Et₂O, tetrachloroethane, 80 °C, 2 h

20.0 equiv BF₃–Et₂O, tetrachloroethane, 80 °C, 16 h

(iii)

9

10

11

14% 74

20% 74

39% 74

O

N

O

N

agents and conditions, comprising Brönsted acids, Lewis acids, and

super acids (Scheme 7). A representative selection of experiments

is listed in Table 2. The use of formic acid as solvent resulted in

isomerisation of the double bond and the oleﬁn 75 was isolated

(entry 1). When the unsaturated alcohol 73 was heated in a mix-

ture of sulfuric acid and acetic acid (1:2), the desired spiro com-

pound 74 was obtained in low yield (entries 2 and 3). Phosphoric

acid was found to be the reagent of choice for the synthesis of race-

mic 7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridines of the

general formula 3.⁵However, in the present case, the alcohol 73

showed little solubility in 85% phosphoric acid and the desired

spiro compound 74 was obtained in low yield only applying a vari-

ety of reaction temperatures (entries 4–6). Experiments aiming to

enhance the solubility of 73 in 85% phosphoric acid by addition of

an organic co-solvent (DMSO) remained inconclusive. No deﬁned

product was obtained, when the substrate 73 was heated in poly-

phosphoric acid (130 °C, 2 h). The cycloisomerisation of 73 in the

presence of aqueous hydrobromic acid and tributylhexadecylphos-

phonium bromide as phase transfer catalyst (110 °C, 2 h) afforded

target compound 74 in 11% yield (entry 7). No reaction occurred,

when the cycloisomerisation of 73 was attempted under milder

conditions, using either methanesulfonic acid (DMSO, room tem-

perature to 80 °C), Montmorillonit K-10 clay (benzene, 50–80 °C),

or hexaﬂuoroisopropanol (50 °C).³⁶The use of ﬂuorosulfuric acid

under conditions described in literature (nitropropane, ꢀ75 °C)

afforded the spiro derivative 74 in low yield only (entry 8).³⁷When

the reaction mixture was warmed to 0 °C, decomposition occurred.

Finally, the cyclisation of 73 was studied in the presence of Le-

wis acids.³⁸Tetrakis(2,2,2-triﬂuoroacetoxy)stannane was synthe-

sized according to the literature procedures.³⁹Cycloisomerisation

of 73–74 took place only in the presence of at least stoichiometric

amounts of tetrakis(2,2,2-triﬂuoroacetoxy)stannane. At a tempera-

ture of 120 °C, the target compound 74 was obtained in 14% yield

(entry 9). The best results were achieved using an excess of boron

triﬂuoride in tetrachloroethane: after a reaction time of 16 h at

80 °C, the spiro derivative 74 was isolated in 39% yield (entry

11). It was demonstrated that no reaction occurred at lower tem-

peratures (room temperature or 50 °C) and that target compound

74 was obtained in lower yield (20%) when the reaction time

was decreased to 2 h (entry 10).

N

(iv)

N

OH

74

73

(v)

65

O

(vi)

N

OH

75

72

Scheme 7. Reagents and conditions: (i) catalyst 15, oleﬁn 65, 1,2-dichloroethane,

80 °C, 2 h (2ꢁ), 44% (ii) catalyst 16, addition of oleﬁn 65 over 4 h, 1,2-dichloroeth-

ane, 80 °C, 4 h, 27%; (iii) LiOH, H₂O, 1,4-dioxane, 100 °C, 1 h, 80%; (iv) see Table 2,

entries 2–11; (v) HCOOH, microwave, 150 °C, 0.5 h, 18%; (vi) catalyst 15, 1,2-

dichloroethane, 80 °C, 2 h, 78%.

As in the asymmetric series, the cross-metathesis reaction with

2-methyleneindane (65) was also studied using the thexyldimeth-

ylsilyl-protected oleﬁn 29. However, in contrast to the asymmetric

series, this did not result in an improved outcome of the reaction.

It was also investigated whether the indane moiety could be

constructed after the cross-metathesis step (Scheme 8). As ex-

pected, the cross-metathesis reaction between 29 and 1-allyl-2-

(chloromethyl)benzene 70 proceeded in a less sluggish manner

(8 mol% of catalyst 16, dichloroethane, 45 °C, 20 h) and the hetero-

dimer 76 was isolated in 72% yield. However, attempted Heck

cyclisation of oleﬁn 76 under the conditions described for the

synthesis of 2-methyleneindane (65), yielded a complex mixture.

The acid-catalyzed cycloisomerisation of the unsaturated alco-

hol 73 to the spiro derivative 74 was studied using a variety of re-

O

(i)

(ii)

x

N

Cl

OTxDMS

29

76

77

Scheme 8. Reagents and conditions: (i) 1-allyl-2-(chloromethyl)benzene 70, catalyst 16, dichloromethane, 45 °C, 20 h, 72%; (ii) Pd(PPh₃)₄, NEt₃, CH₃CN, 85 °C, 3 h, no deﬁned

product.

374

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

Table 3

Inhibitory activity and physicochemical properties of spiro derivatives 33, 35, 36, 46–55, 62, 63, 74 and reference compounds 6, 7, 34.

O

R3

N

O

R1

R2

6 (BYK 311319)

7

74

34

33-36, 46-55, 62-63

Compound

R1

R2

R3

H⁺/K⁺-ATPase—log IC₅₀

Gastric Glands—log IC₅₀

pK_a

logD (pH 7.4)

34 (rac.)

6 (9S)-ent.

7 (9R)-ent.

5.8

6.8

5.4

6.3

6.7

5.6

7.15

2.83

O

33 (rac.)

35 (ent. 1)

36 (ent. 2)

46

H

5.8

5.5

5.9

6.2

6.6

6.2

6.6

6.84

2.91

N

O

6.80

6.76

3.29

3.64

N

H

O

N

H

47

H

6.4

O

48

49

H

6.4

6.0

6.9

6.4

6.81

6.65

3.19

3.17

N

O

F

N

F

50

51

52

H

5.3

5.8

5.3

6.4

6.7

6.6

6.55

6.88

6.81

3.57

3.36

3.87

N

O

N

O

N

O

53

54

H

5.4

6.6

6.0

6.6

6.74

6.76

2.85

4.70

O

N

O

N

H

O

55

62

63

H

F

H

6.3

6.2

6.7

7.0

6.63

n. d.

3.15

3.0^a

N

O

H

N

O

H

CH₃

5.6

6.5

6.3

6.1

6.87

6.84

3.42

2.9^a

N

74

a

logD value determined by HPLC.

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

375

2.2. Biological and physicochemical evaluation of

spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes)

dane moiety. As can be seen from the data presented in Table 3,

ﬂuoro-substitution not only was tolerated, but furnished the most

active inhibitor in the gastric glands model. Incorporation of a

methyl substituent in the indane moiety resulted in a slight de-

crease of activity (as compared to parent compound 33) and a pro-

nounced increase in lipophilicity (logD value of 3.42 vs 2.91).

Also, the only inhibitor synthesized in the symmetric spiro(imi-

The inhibitory and cellular activity of the spiro-substituted 7H-

8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridines 33, 35, 36, 46–

55, 62, 63 and 74 was evaluated in a competitive binding assay

against H⁺/K⁺-ATPase from hog gastric mucosa and by determina-

tion of acid formation in gastric glands. Additionally, the lipophil-

icity and pK_avalues of these target compounds were determined.

The results are summarized in Table 3.

dazo[1,2-a]pyrano[2,3-c]pyridine-9,2⁰-indene)

series

showed

promising biological activity: in comparison to its analogue 33

from the asymmetric series, compound 74 inhibited the H⁺/K⁺-

ATPase in a more potent manner (pIC₅₀= 6.5 vs 5.8). On the other

hand, the asymmetric derivative 33 was more active in the gastric

glands model (pIC₅₀= 6.6 vs 6.1). Both compounds possessed com-

parable physicochemical data (pK_avalue and lipophilicity).

To our delight, spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-

indene) 33 turned out to be a potent inhibitor of the gastric proton

pump enzyme comparable with the already known analogous 9-

phenyl-7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridine

34.

Both derivatives showed a pIC₅₀value of 5.8. Spiro derivative 33

inhibited the ¹⁴C-dimethylaminopyridine accumulation in gastric

glands in an even more pronounced manner than reference com-

pound 34 (pIC₅₀= 6.6 vs 6.3). Both compounds (33 and 34) showed

a comparable physicochemical proﬁle with favourable pK_avalues

(6.84/7.15) and lipophilicity (logD = 2.91/2.83). Interestingly, in

the spiro series both enantiomers 35 and 36 contributed to the

inhibitory activity of the racemic mixture 33, possessed compara-

ble afﬁnity towards the H⁺/K⁺-ATPase (pIC₅₀= 5.5 vs 5.9), and

exhibited comparable activity in the gastric glands model

(pIC₅₀= 6.2 vs 6.6). In the case of the 9-aryl-7H-8,9-dihydropyr-

ano[2,3-c]imidazo[1,2-a]pyridine series 3, the (9S)-enantiomers 4

showed signiﬁcantly higher biological activity than their (9R)-

antipodes 5 (cf. Table 3: 6 vs 7).

In summary, compounds belonging to both classes 8 and 9

showed promising inhibitory activity and constitute promising

candidates for further development as anti-ulcer drugs. Especially

spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes) 33, 46, 48,

55, 62 (asymmetric series) and 74 (symmetric series) possess a bal-

anced proﬁle of favourable biochemical activity and promising

physicochemical properties. The ﬁnding that spiro(imidazo[1,2-

a]pyrano[2,3-c]pyridine-9-indenes) constitute potent inhibitors of

the gastric proton pump enzyme is noteworthy, since they repre-

sent the ﬁrst examples for P-CABs, in which the distance between

the heterocyclic scaffold and the aryl residue has been modiﬁed.

3. Conclusion

Potent inhibition of the H⁺/K⁺-ATPase and good activity in the

gastric glands model was observed for a variety of 6-carboxamide

A series of asymmetric and symmetric spiro(imidazo[1,2-a]pyr-

ano[2,3-c]pyridine-9-indenes) 8 and 9 was prepared from readily

available 7-allyl-imidazo[1,2-a]pyridine building blocks (28, 29

and 40) and methyleneindanes (23–25 and 65) by cross-metathe-

sis reaction and subsequent acid-catalyzed cycloisomerisation.

Although second-generation Grubbs catalyst 15 showed higher

activity, the use of Hoveyda–Grubbs catalyst 16 was preferred,

since it afforded the target compounds in higher purity. In the

asymmetric series, the cyclisation reaction proceeded smoothly

in the presence of 85% phosphoric acid. The cyloisomerisation

was found to be more challenging in the symmetric series and

the Lewis acid boron triﬂuoride etherate was identiﬁed as the re-

agent of choice. Despite the fact that—in comparison to known P-

CABs—the distance between the heterocyclic scaffold and the aryl

residue has been modiﬁed, the target compounds showed promis-

ing inhibitory activity. Due to their balanced proﬁle of favourable

biochemical activity and promising physicochemical properties,

the inhibitors 33, 46, 48, 55, 62 and 74 constitute promising candi-

dates for further development as anti-ulcer drugs.

substituted

spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-ind-

enes) 8: the monosubstituted amides 46 and 47 possessing a

methyl/cyclopropyl residue, respectively, were highly active com-

pounds and possessed pK_avalues comparable to parent compound

33. However, both derivatives were found to be more lipophilic

than dimethylcarboxamide 33. The increase in lipophilicity

(logD = 4.70) was even more pronounced in the case of phenyl-

carboxamide 54, which constituted the most active compound in

this series (pIC₅₀= 6.6). On the other hand, both, the azetidine car-

boxamide 48 and the Weinreb amide 55 were found to be only

slightly more lipophilic than parent compound 33 (logD values

of 3.19/3.15 vs 2.91). Both compounds 48 and 55 showed good bio-

logical activity, especially in the gastric glands model, where pIC₅₀

values of 6.9 and 6.7 were determined. Incorporation of one or two

ﬂuoro atoms into 3-position of the azetidine moiety (compounds

49 and 50) seemed to compromise the afﬁnity towards the gastric

proton pump enzyme (decrease of pIC₅₀from 6.4 to 6.0/5.3, respec-

tively) and, in a less pronounced manner, the activity in gastric

glands (reduction of pIC₅₀from 6.9 to 6.4). Whereas the presence

of ﬂuoro substituents inﬂuenced the pK_avalue of 49 and 50 in a

favourable manner (decrease to values of 6.65/6.55, respectively),

an undesired increase in lipophilicity (logD = 3.57) was observed

for diﬂuoro derivative 50. Likewise, the increase of the ring size

led to a reduction of afﬁnity towards H⁺/K⁺-ATPase and an increase

in lipophilicity (cf. pyrrolidine carboxamide 51 and piperidine car-

boxamide 52). Interestingly, the less potent enzyme inhibition of

target compounds 51 and 52 was not reﬂected in the reduction

of the ¹⁴C-dimethylaminopyridine accumulation in gastric glands,

where high pIC₅₀values of 6.7 and 6.6 were observed. Replacement

of the piperidine moiety versus a morpholine moiety resulted in a

pronounced decrease of lipophilicity of more than one log unit (cf.

52 vs 53). Despite its favourable lipophilicity, morpholine carbox-

amide 53 was not evaluated in more detail, since it represented

one of the weakest inhibitors listed in Table 3.

4. Experimental

4.1. Chemistry

4.1.1. General

All chemicals were purchased from the major chemical suppli-

ers as highest purity grade and used without any further puriﬁca-

tion. Compounds 27, 29 and 38 were prepared as described

previously.⁵The progress of the reaction was monitored on Mache-

rey-Nagel HPTLC plates Nano-SIL 20 UV₂₅₄(0.20 mm layer, nano-

silica gel 60 with ﬂuorescence indicator UV₂₅₄) using dichloro-

methane/methanol as solvent system. Column chromatography

was performed with Merck silica gel 60 (70–230 mesh ASTM) with

the solvent mixtures speciﬁed in the corresponding experiment.

Spots were visualized by iodine vapour or by irradiation with ultra-

violet light (254 nm). Melting points (mp) were taken in open cap-

Different results were obtained for the two target compounds

62 and 63, which possessed a ﬂuoro-/methyl-substituent in the in-

illaries on

a Büchi B-540 melting point apparatus and are

376

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

uncorrected. ¹H NMR spectra were recorded with a Bruker DRX

200 FT-NMR spectrometer at a frequency of 200.1 MHz or a Bruker

AV 400 FT-NMR spectrometer at a frequency of 400.1 MHz. CDCl₃

or DMSO-d₆was used as solvents. The chemical shifts were re-

ported as parts per million (d ppm) with tetramethylsilane (TMS)

as an internal standard. High resolution mass spectra were ob-

tained on a Bruker Daltonics MicroTOF Focus instrument using

electrospray ionisation (ESI positive). Elemental analysis was per-

formed on a Carlo Erba 1106 C, H, N analyzer.

gel, eluant: petrol ether/ethyl acetate = 9:1 (v/v), then ethyl ace-

tate] afforded the title compound in 25% yield (450 mg of a beige

foam): ¹H NMR (CDCl₃, 200 MHz): d = 1.48 (s, 9H), 2.34, 2.39 (2s,

6H), 2.79 (s, m_c, 5H), 2.94 (s, 3H), 3.02 (m_c, 2H), 3.53 (m_c, 2H),

5.83 (m_c, 1H), 7.12–7.36 (m), 7.57 (s, 1H).

4.1.5. 7-[(2E)-2-(2,3-Dihydro-1H-inden-1-ylidene)ethyl]-8-

{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}-N,N,2,3-tetra-

methylimidazo[1,2-a]pyridine-6-carboxamide (31)

In a ﬂask ﬁlled with argon, 1-methyleneindane 23 (1.90 g,

14.6 mmol) and catalyst 15 (380 mg, 0.45 mmol) was added to a

solution of 29 (2.00 g, 4.8 mmol) in dry dichloromethane (10 ml).

The reaction mixture was stirred for 18 h at 40 °C and concen-

trated. Puriﬁcation by column chromatography [100 g of silica

gel, eluant: petrol ether, then petrol ether/ethyl acetate = 4:6

(v/v)] afforded the title compound in 72% yield (2.0 g of a greenish

foam): ¹H NMR (CDCl₃, 200 MHz): d = 0.43 (br s, 6H), 0.97 (d, 6H),

1.03 (s, 6H), 1.88 (m_c, 1H), 2.31, 2.37 (2s, 6H), 2.71, 2.72 (s, m_c, 5H),

2.94, 2.99 (s, m_c, 5H), 3.69 (br s, 2H), 5.90 (m_c, 1H), 7.11–7.32 (m).

4.1.2. Synthesis of 1-methyleneindanes 23–25 from 1-indanones

20–22

At a temperature of 0 °C, methyltriphenylphosphonium bro-

mide (1.1 equiv) and potassium tert-butylate (1.1 equiv) was

added to a solution of the corresponding indanone in diethyl ether.

The suspension was stirred for 2–20 h at room temperature. The

solvent was evaporated and the residue extracted with n-pentane

(3–5ꢁ). The combined extracts were evaporated to dryness. The

corresponding title compound was puriﬁed by vacuum distillation

or by column chromatography.

4.1.6. 7-[(2E)-2-(2,3-Dihydro-1H-inden-1-ylidene)ethyl]-8-

hydroxy-N,N,2,3-tetramethylimidazo[1,2-a]pyridine-6-

carboxamide (32)

4.1.2.1. 1-Methyleneindane (23). The title compound was ob-

tained from 1-indanone 20 (20.0 g, 151 mmol) as described in

the general procedure (12.0 g of a colourless oil, 61% yield): bp

39 °C/0.9 mbar.

4.1.6.1. Synthesis of 32 by cleavage of the pivaloyl protecting

group. A solution of 30 (450 mg, 0.98 mmol) in 1,4-dioxane

(15 ml) was treated with 3 N aqueous lithium hydroxide solution

(1.2 ml) and heated for 30 min at 100 °C. After cooling to room

temperature, saturated ammonium chloride solution (20 ml) and

dichloromethane (30 ml) was added. The phases were separated

and the aqueous phase was extracted with dichloromethane

(10 ml). The combined organic phases were dried over sodium sul-

fate and concentrated under reduced pressure. The oily residue

(0.5 g) was puriﬁed by column chromatography [15 g of silica

gel, eluant: ethyl acetate]. Evaporation of the corresponding frac-

tions afforded the title compound (240 mg of a greenish solid,

66% yield): ¹H NMR (CDCl₃, 200 MHz): d = 2.31, 2.38 (2s, 6H),

2.80–2.90, 2.80 (m_c, s, 5H), 2.98–3.09, 3.03 (m_c, s, 5H), 3.74 (br s,

2H), 6.04 (m_c, 1H), 7.10–7.38 (m).

4.1.2.2. 5-Fluoro-1-methyleneindane (24). The title compound

was obtained from 5-ﬂuoro-1-indanone 21 (5.0 g, 33.3 mmol) as

described in the general procedure [4.17 g of a yellow oil, 85%

yield, eluant for column chromatography: petrol ether/ethyl ace-

tate/triethylamine = 8:1:1 (v/v/v)]: ¹H NMR (C₆D₆, 200 MHz):

d = 2.55 (s, 4H), 5.01 (d, 1H), 5.40 (d, 1H), 6.80 (m_c, 2H), 7.22 (m_c).

4.1.2.3. 4-Methyl-1-methyleneindane (25). The title compound

was obtained from 4-methyl-1-indanone 22 (5.0 g, 34.2 mmol) as

described in the general procedure [4.77 g of a brown oil, 97% yield,

eluant for column chromatography: petrol ether/ethyl acetate/tri-

ethylamine = 8:1:1 (v/v/v)]: ¹H NMR (C₆D₆, 200 MHz): d = 2.00 (s,

3H), 2.55 (m_c, 4H), 5.03 (m_c, 1H), 5.50 (m_c, 1H), 6.91 (d, 1H), 7.05

(t, 2H), 7.34 (d, 1H).

4.1.6.2. Synthesis of 32 by cleavage of the [dimethyl(1,1,2-

trimethylpropyl)silyl] protecting group. Tetrabutylammonium

ﬂuoride (1 M solution in THF, 5.20 ml, 5.2 mmol) was added to

a solution of 31 (1.80 g, 3.5 mmol) in THF (25 ml). The reaction

mixture was stirred for 1 h at room temperature and quenched

with saturated ammonium chloride solution (50 ml) and dichlo-

romethane (80 ml). The phases were separated and the aqueous

phase extracted with dichloromethane (2ꢁ 20 ml). The combined

organic phases were dried over sodium sulfate and concentrated

under reduced pressure. The residue (3.9 g of a green oil) was

puriﬁed by column chromatography [110 g of silica gel, eluant:

ethyl acetate]. This afforded 2.3 g of a mixture of the title com-

pound (32 mol%, 41 wt%, 71% corrected yield) and tetrabutylam-

monium hydroxide (68 mol%, 59 wt%): ¹H NMR (CDCl₃,

200 MHz): d = 2.31, 2.38 (2s, 6H), 2.78–2.91, 2.80 (m_c, s, 5H),

2.91–3.10, 3.03 (m_c, s, 5H), 3.73 (br s, 2H), 6.03 (m_c, 1H),

7.10–7.35 (m), signals of tetrabutylammonium hydroxide: 1.01

(t), 1.46 (m_c), 1.69 (m_c), 3.40 (m_c).

4.1.3. 7-Allyl-6-(dimethylcarbamoyl)-2,3-dimethylimidazo[1,2-

a]pyridin-8-yl pivalate (28)

A suspension of 27 (1.50 g, 5.5 mmol) in acetone (60 ml) was

treated with potassium carbonate (0.76 g, 5.5 mmol) and pivaloyl

chloride (1.33 g, 11.0 mmol). After a period of 45 min at room tem-

perature, the reaction mixture was quenched by addition of satu-

rated ammonium chloride solution (50 ml) and dichloromethane

(120 ml). The phases were separated and the aqueous phase was

extracted with dichloromethane (2ꢁ 50 ml). The combined organic

phases were dried over sodium sulfate and concentrated under re-

duced pressure. The crude product (2.5 g of a beige solid) was puri-

ﬁed by column chromatography (75 g of silica gel, eluant: ethyl

acetate). Evaporation of the corresponding fractions afforded the

title compound (1.4 g of a colourless solid, 71% yield). ¹H NMR

(CDCl₃, 200 MHz): d = 1.48 (s, 9H), 2.35, 2.39 (2s, 6H), 2.90, 3.11

(2s, 6H), 3.35 (m_c, 2H), 5.04 (m_c, 1H), 5.79 (m_c, 1H), 7.61 (s, 1H).

4.1.4. 7-[(2E)-2-(2,3-Dihydro-1H-inden-1-ylidene)ethyl]-6-

(dimethylcarbamoyl)-2,3-dimethyl-imidazo[1,2-a]pyridin-8-yl

pivalate (30)

In a ﬂask ﬁlled with argon, 1-methyleneindane 23 (1.90 g,

14.6 mmol) and catalyst 15 (266 mg, 0.31 mmol) was added to a

solution of 28 (1.40 g, 3.9 mmol) in dry 1,2-dichloroethane

(50 ml). The reaction mixture was stirred for 2 h at 80 °C and con-

centrated. Puriﬁcation by column chromatography [150 g of silica

4.1.7. N,N,2,3-Tetramethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo

[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide (33)

A suspension of 32 (2.30 g, mixture with tetrabutylammonium

hydroxide, 41 wt%, 3.6 mmol) in 85% phosphoric acid (7 ml) was

heated at 50 °C (pre-heated oil bath). After a period of 10 min, a

solution was obtained. Stirring was continued for 50 min. The reac-

tion mixture was poured on ice and diluted with dichloromethane

(80 ml). A pH value of 8 was adjusted by addition of 6 N sodium

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

377

hydroxide solution. The phases were separated and the aqueous

phase was extracted with dichloromethane (2ꢁ 10 ml). The com-

bined organic phases were dried over sodium sulfate and the sol-

vent was evaporated in vacuo. The crude product (1.5 g of a

green oil) was puriﬁed by column chromatography [60 g of silica

gel, eluant: ethyl acetate/methanol = 9:1 (v/v)]. Evaporation of

the corresponding fractions afforded the title compound (0.95 g

of a beige foam, 70% corrected yield). In cases, where the respective

batch of 33 contained traces of by-product 34, the title compound

was further puriﬁed by preparative HPLC: ¹H NMR (DMSO-d₆,

200 MHz): d = 2.06 (m_c, 1H), 2.20, 2.23 (s, m_c, 5H), 2.33, 2.38 (s,

m_c, 4H), 2.71 (m_c, 2H), 2.92, 2.98, 3.03 (s, m_c, s, 8H), 7.34 (m_c,

4H), 7.77 (s, 1H); HRMS (ESI) m/z C₂₃H₂₆N₃O₂[M+H]⁺Calcd:

376.2020. Found: 376.2019.

ant: petrol ether/ethyl acetate = 1:1 (v/v), then ethyl acetate]. This

afforded 5.7 g of ethyl 8-(allyloxy)-2,3-dimethylimidazo[1,2-a]pyr-

idine-6-carboxylate (yellow solid, 47% yield): ¹H NMR (CDCl₃,

200 MHz): d = 1.42 (t, 3H), 2.43 (s, 6H), 4.41 (q, 2H), 4.81 (d, 2H),

5.33 (dd, 1H), 5.47 (dd, 1H), 6.17 (ddd, 1H), 6.99 (d, 1H), 8.26 (d,

1H); HRMS (ESI) m/z C₁₅H₁₉N₂O₃[M+H]⁺Calcd: 275.1390. Found:

275.1376. A ﬂask containing 22 g (80 mmol) of ethyl 8-(allyloxy)-

2,3-dimethylimidazo[1,2-a]pyridine-6-carboxylate was heated for

1.5 h at 160 °C. The material melted after a period of 5 min and

solidiﬁed after 45 min. The crude product was dissolved in acetone

(40 ml) and diethyl ether (30 ml). After a period of 30 min at room

temperature, the precipitate was isolated by ﬁltration and washed

with diethyl ether (20 ml). This afforded 16.2 g of the title com-

pound (off-white solid, 74% yield). The mother liquor was concen-

trated and the brown oily residue (12 g) was puriﬁed by column

chromatography [400 g of silica gel, eluant: dichloromethane/

methanol = 100:3 (v/v)]. Another 1.6 g (7% yield) of the title com-

pound was isolated: ¹H NMR (CDCl₃, 200 MHz): d = 1.42 (t, 3H),

2.39 (s, 6H), 3.94 (d, 2H), 4.39 (q, 2H), 5.02 (m_c, 2H), 6.06 (ddd,

1H), 8.09 (s, 1H); HRMS (ESI) m/z C₁₅H₁₉N₂O₃[M+H]⁺Calcd:

275.1390. Found: 275.1379.

4.1.8. (ꢀ)-N,N,2,3-Tetramethyl-2⁰,3⁰,7,8-tetrahydrospiro[imi-

dazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide

(35) and (+)-N,N,2,3-Tetramethyl-2⁰,3⁰,7,8-tetrahydrospiro

[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carbo-

xamide (36)

Resolution of 33 (1.30 g, 3.5 mmol) was achieved by preparative

chromatography using a 250 ꢁ 30 mm CHIRALPAK^ÒAD-H 5

lm

column. The mobile phase consisted of a mixture of n-heptane, iso-

propanol and diethylamine [80:20:0.1 (v/v/v)]. The separation was

performed at room temperature with a ﬂow rate of 42.5 ml/min.

The products were detected at a wavelength of 230 nm. The (ꢀ)-

enantiomer (611 mg, 47% yield, 99.4% ee) was eluted ﬁrst: mp

4.1.11. Ethyl 7-allyl-8-{[dimethyl(1,1,2-trimethylpropyl)

silyl]oxy}-2,3-dimethylimidazo[1,2-a]pyridine-6-carboxylate (40)

4.1.11.1. Synthesis of 40 using triethylamine as base. A solution

of 39 (1.30 g, 4.7 mmol) and triethylamine (0.95 g, 9.4 mmol) in

dichloromethane

(30 ml)

was

cooled

to

0 °C

and

211–212 °C; optical rotation: ½a ₂^D₀

ꢂ

ꢀ45° (c 0.54, chloroform); ¹H

chloro(dimethyl)thexylsilane (1.70 g, 9.5 mmol) added. The reac-

tion mixture was stirred for 18 h at room temperature and

quenched with saturated ammonium chloride solution (30 ml)

and dichloromethane (20 ml). The phases were separated and the

aqueous phase was extracted with dichloromethane (2ꢁ 10 ml).

The combined organic phases were dried over sodium sulfate

and concentrated in vacuo. The residue (3.1 g of a brown oil) was

puriﬁed by column chromatography [90 g of silica gel, eluant: pet-

rol ether/ethyl acetate = 10:1 (v/v)]. This afforded 2.3 g of a yellow

solid [mixture of the title compound (45 mol%, 68 wt%) and 41

(55 mol%, 32 wt%), 80% corrected yield]: ¹H NMR (CDCl₃,

200 MHz): d = 0.38 (s, 6H), 0.97 (d, 6H), 1.03 (s, 6H), 1.39 (t, 3H),

1.88 (m_c, 1H), 2.37 (s, 6H), 3.87 (m_c, 2H), 4.35 (q, 2H), 4.89 (m_c,

2H), 5.93 (ddd, 1H), 8.14 (s, 1H), signals of 41: 0.14 (s, 6H), 0.87

(s, 6H), 0.91 (d, 6H), 1.64 (m_c, 1H).

NMR (200 MHz, DMSO-d₆): d = 2.06 (m_c, 1H), 2.20, 2.23 (s, m_c,

5H), 2.33, 2.38 (s, m_c, 4H), 2.71 (m_c, 2H), 2.92, 2.98, 3.03 (s, m_c, s,

8H), 7.34 (m_c, 4H), 7.77 (s, 1H); HRMS (ESI) m/z C₂₃H₂₆N₃O₂

[M+H]⁺Calcd: 376.2020. Found: 376.2019. The (+)-enantiomer

(569 mg, 44% yield, 99.8% ee) was eluted next: mp 216–217 °C;

optical rotation:

½

a ^D₂₀45° (c 0.44, chloroform); ¹H NMR

ꢂ

(200 MHz, DMSO-d₆): d = 2.06 (m_c, 1H), 2.20, 2.23 (s, m_c, 5H),

2.33, 2.38 (s, m_c, 4H), 2.71 (m_c, 2H), 2.92, 2.98, 3.03 (s, m_c, s, 8H),

7.34 (m_c, 4H), 7.77 (s, 1H); HRMS (ESI) m/z C₂₃H₂₆N₃O₂[M+H]⁺

Calcd: 376.2020. Found: 376.2017. Analytical HPLC method for

the determination of the optical purity: column: 250 ꢁ 4.6 mm

CHIRALPAK^ÒAD-H; mobile phase: n-heptane/isopropanol [80:20

(v/v)]; ﬂow rate: 1 ml/min; room temperature; detection at

245 nm: t_R[(ꢀ)-enantiomer] = 9.3 min; t_R[(+)-enantiomer] = 14.3 min.

4.1.9. N,N,2,3-Tetramethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo[1,2-

a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide (2Z)-but-2-

enedioate (37)

Compound 33 (200 mg, 0.53 mmol) was dissolved in acetone

(2 ml) and maleic acid (60 mg, 0.52 mmol) was added. A precipi-

tate was formed which was isolated by ﬁltration and washed with

diethyl ether. The title compound was isolated in 73% yield

(190 mg of a colourless solid): mp 217–218 °C; ¹H NMR (DMSO-

d₆, 200 MHz): d = 2.11, 2.31, 2.42 (m_c, 2s, 10H), 2.83, 2.93, 2.99,

3.07 (m_c, s, m_c, s, 10H), 6.06 (s, 2H), 7.40 (m_c, 4H), 8.25 (s, 1H).

4.1.11.2. Synthesis of 40 using sodium hydride as base. Under

an argon atmosphere, sodium hydride (1.46 g, 60 wt% in

mineral oil, 36.5 mmol) was washed with dry THF (2ꢁ 20 ml)

and added to a solution of 39 (5.00 g, 18.2 mmol) in dichloro-

methane (100 ml). The solution was stirred for 30 min and

chloro(dimethyl)thexylsilane (4.03 ml, 3.67 g, 20.5 mmol) added

rapidly. The reaction mixture was stirred for 2 h at room temper-

ature and quenched with saturated ammonium chloride solution

(100 ml) and water (50 ml). The phases were separated and the

aqueous phase was extracted with dichloromethane (2ꢁ 50 ml).

The combined organic phases were dried over magnesium sulfate

and concentrated in vacuo. The residue (a brown oil) was puriﬁed

by column chromatography [200 g of silica gel, eluant: petrol

ether/ethyl acetate = 10:1 (v/v)]. This afforded 6.3 g of a yellow

solid [mixture of the title compound (77 mol%, 90 wt%) and 41

(23 mol%, 10 wt%), 74% corrected yield]: ¹H NMR (DMSO-d₆,

200 MHz): d = 0.34 (s, 6H), 0.93 (d, 6H), 0.99 (s, 6H), 1.32 (t,

3H), 1.82 (m_c, 1H), 2.31, 2.40 (2s, 6H), 3.78 (m_c, 2H), 4.28 (q,

2H), 4.78 (dd, 1H), 4.93 (dd, 1H), 5.87 (ddd, 1H), 8.27 (s, 1H), sig-

nals of 41: 0.00 (s, 6H), 0.79 (s, 6H), 0.85 (d, 6H), 1.57 (m_c, 1H);

HRMS (ESI) m/z C₂₃H₃₇N₂O₃Si [M+H]⁺Calcd: 417.2568. Found:

417.2571.

4.1.10. Ethyl 7-allyl-8-hydroxy-2,3-dimethylimidazo[1,2-a]

pyridine-6-carboxylate (39)

Allyl bromide (9.7 g, 80 mmol) and potassium carbonate (9.2 g,

67 mmol) was added to a suspension of 38 (10.4 g, 44 mmol) in dry

DMF (250 ml). The reaction mixture was stirred for 2.5 h at room

temperature and concentrated under reduced pressure. The resi-

due was treated with dichloromethane (200 ml) and saturated

ammonium chloride solution (100 ml). The phases were separated

and the aqueous phase was extracted with dichloromethane (2ꢁ

50 ml). The combined organic phases were dried over sodium sul-

fate and evaporated to dryness. The brown oily residue (14.3 g)

was puriﬁed by column chromatography [400 g of silica gel, elu-

378

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

4.1.12. Ethyl 7-[(2E)-2-(2,3-dihydro-1H-inden-1-ylidene)ethyl]-

8-{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}-2,3-dimethyl-

imidazo[1,2-a]pyridine-6-carboxylate (42)

fractions afforded the title compound (0.4 g of an off-white solid,

80% yield): analytical data cf. Section 4.1.14.2.

4.1.12.1. Synthesis of 42 using second-generation Grubbs cata-

lyst 15. In a ﬂask ﬁlled with argon, 1-methyleneindane 23 (1.90 g,

14.6 mmol) and catalyst 15 (380 mg, 0.45 mmol) was added to a

solution of 40 (2.30 g, mixture with 41, 68 wt%, 3.8 mmol) in dry

dichloromethane (10 ml). The reaction mixture was stirred for

18 h at 40 °C and concentrated. Puriﬁcation by column chromatog-

raphy [silica gel, eluant: petrol ether, then petrol ether/ethyl ace-

tate = 10:1 (v/v)] afforded 2.0 g of a beige solid [mixture of the

title compound (38 mol%, 67 wt%) and 41 (62 mol%, 33 wt%), 68%

corrected yield]: ¹H NMR (CDCl₃, 200 MHz): d = 0.39 (s, 6H), 0.96

(d, 6H), 1.04 (s, 6H), 1.30 (t, 3H), 1.89 (m_c, 1H), 2.37 (s, 6H), 2.78

(m_c, 2H), 2.98 (m_c, 2H), 2.97 (d, 2H), 4.31 (q, 2H), 5.91 (m_c, 1H),

7.20 (m_c), 8.09 (s, 1H), signals of 41: 0.14 (s, 6H), 0.87 (s, 6H),

0.91 (d, 6H), 1.64 (m_c, 1H); HRMS (ESI) m/z C₃₁H₄₃N₂O₃Si [M+H]⁺

Calcd: 519.3037. Found: 519.3020.

4.1.14.2. Synthesis of 44 by one-pot deprotection/cyclisation of

42. A suspension of 42 (0.50 g, 66 wt%, 0.64 mmol) in 85% phos-

phoric acid (5 ml) was heated at 50 °C (pre-heated oil bath). After

a period of 30 min, a solution was obtained. Stirring was continued

for 1 h. The reaction mixture was poured on ice and diluted with

dichloromethane (30 ml). A pH value of 8 was adjusted by addition

of 6 N sodium hydroxide solution. The phases were separated and

the aqueous phase was extracted with dichloromethane (2ꢁ

10 ml). The combined organic phases were dried over sodium sul-

fate and the solvent was evaporated in vacuo. The crude product

(0.8 g of a green foam) was puriﬁed by column chromatography

[32 g of silica gel, eluant: ethyl acetate/petrol ether = 6:4 (v/v)].

Evaporation of the corresponding fractions afforded the title com-

pound (0.4 g of a slightly green solid, 91% corrected yield): ¹H NMR

(CDCl₃, 200 MHz): d = 1.44 (t, 3H), 2.04–2.57, 2.38, 2.41 (m, 2s,

10H), 2.92 (m_c, 1H), 3.21 (m_c, 3H), 4.41 (q, 2H), 7.28 (m_c), 8.22 (s,

1H); HRMS (ESI) m/z C₂₃H₂₅N₂O₃[M+H]⁺Calcd: 377.1860. Found:

377.1843.

4.1.12.2. Synthesis of 42 using Hoveyda–Grubbs catalyst 16. In a

ﬂask ﬁlled with argon, 1-methyleneindane 23 (4.74 g, 36.4 mmol)

and catalyst 16 (570 mg, 0.91 mmol) was added to a solution of

40 (8.30 g, mixture with 41, 82 wt%, 16.3 mmol) in dry dichloro-

methane (40 ml). The reaction mixture was stirred for 18 h at

40 °C. Another portion of catalyst 16 (170 mg, 0.27 mmol) was

added and stirring was continued for 6 h. The solvent was evapo-

rated and the crude product puriﬁed by column chromatography

[150 g of silica gel, eluant: petrol ether, then petrol ether/ethyl ace-

tate = 9:1 (v/v)]. Evaporation of the corresponding fractions affor-

ded 7.65 g of a beige solid [mixture of the title compound

(50 wt%), 40 (40 wt%), and 41 (10 wt%), 45% corrected yield]: ana-

lytical data cf. Section 4.1.12.1.

4.1.15. 2,3-Dimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo[1,2-a]-

pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxylic acid (45)

A suspension of 44 (2.20 g, 5.8 mmol) in ethanol (20 ml) and 6 N

potassium hydroxide solution (5 ml) was stirred for 30 min at

60 °C. A yellow solution was obtained. At room temperature, a

pH value of 5–6 was adjusted by addition of 4 N hydrochloric acid.

The mixture was diluted with dichloromethane and water. The

phases were separated. The aqueous phase was extracted with

dichloromethane (3ꢁ). The combined organic phases were dried

over sodium sulfate and concentrated under reduced pressure.

The solid residue was suspended in diethyl ether. The title com-

pound was isolated by ﬁltration (2.0 g of a colourless solid, 98%

yield): mp 243–244 °C (decomp.); ¹H NMR (DMSO-d₆, 200 MHz):

d = 2.08 (m_c, 1H), 2.15–2.30, 2.21, 2.37 (m, 2s, 9H), 2.88–3.21 (m,

4H), 7.38 (m_c, 4H), 8.25 (s, 1H); HRMS (ESI) m/z C₂₁H₂₁N₂O₃

[M+H]⁺Calcd: 349.1547. Found: 349.1539.

4.1.13. Ethyl 7-[(2E)-2-(2,3-dihydro-1H-inden-1-ylidene)ethyl]-

8-hydroxy-2,3-dimethylimidazo[1,2-a]pyridine-6-carboxylate (43)

Tetrabutylammonium ﬂuoride (1 M solution in THF, 5.80 ml,

5.8 mmol) was added to a solution of 42 (2.0 g, mixture with 41,

67 wt%, 2.6 mmol) in THF (25 ml). The reaction mixture was stirred

for 1 h at room temperature and quenched with saturated ammo-

nium chloride solution (50 ml) and dichloromethane (80 ml). The

phases were separated and the aqueous phase was extracted with

dichloromethane (2ꢁ 20 ml). The combined organic phases were

dried over sodium sulfate and concentrated under reduced pres-

sure. The residue (3.8 g of a green oil) was puriﬁed by column chro-

matography [110 g of silica gel, eluant: ethyl acetate/petrol

ether = 9:1 (v/v)]. The pure title compound was isolated in 52% cor-

rected yield (0.5 g of a slightly green solid): ¹H NMR (CDCl₃,

200 MHz): d = 1.39 (t, 3H), 2.35, 2.37 (2s, 6H), 2.98 (m_c, 4H), 4.05

(d, 2H), 4.39 (q, 2H), 6.07 (t, 1H), 7.16 (m_c, 3H), 7.35 (m_c, 1H),

8.07 (s, 1H); HRMS (ESI) m/z C₂₃H₂₄N₂O₃[M+H]⁺Calcd:

377.1860. Found: 377.1850.

4.1.16. General procedure for the synthesis of amides 46–55

from carboxylic acid 45

A solution of carboxylic acid 45, O-benzotriazol-1-yl-N,N,N⁰,N⁰-

tetramethyluronium tetraﬂuoroborate (TBTU, 1.5 equiv) and N-

ethyldiisopropylamine (2.5 equiv) in DMF was stirred for 1 h at

40 °C. At room temperature, the corresponding amine (2.0 equiv)

was added to the orange-brown solution and stirring was contin-

ued for 1–3 h. The reaction mixture was concentrated and the res-

idue dissolved in dichloromethane and water. The phases were

separated. The aqueous phase was extracted with dichloromethane

(2–3ꢁ). The combined organic phases were dried over sodium sul-

fate and the solvent was evaporated. The residue was puriﬁed by

column chromatography. Evaporation of the corresponding frac-

tions afforded the title compound, which was further puriﬁed by

crystallization from a suitable solvent or washing with a suitable

solvent. If second-generation Grubbs catalyst 15 was used for the

synthesis of the batch of carboxylic acid 45 employed for amide

coupling, the obtained amide was puriﬁed by preparative HPLC

to remove the respective by-product of the general formula 3.

4.1.14. Ethyl 2,3-dimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo-[1,2-

a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxylate (44)

4.1.14.1. Synthesis of 44 by cyclisation of 43. A suspension of 43

(0.50 g, 1.3 mmol) in 85% phosphoric acid (5 ml) was heated at

50 °C (pre-heated oil bath). After a period of 10 min, a solution

was obtained. Stirring was continued for 50 min. The reaction mix-

ture was poured on ice and diluted with dichloromethane (30 ml).

A pH value of 8 was adjusted by addition of 6 N sodium hydroxide

solution. The phases were separated and the aqueous phase was

extracted with dichloromethane (2ꢁ 10 ml). The combined organic

phases were dried over sodium sulfate and the solvent was evapo-

rated in vacuo. The crude product (0.8 g of a brown foam) was puri-

ﬁed by column chromatography [24 g of silica gel, eluant: ethyl

acetate/petrol ether = 6:4 (v/v)]. Evaporation of the corresponding

4.1.16.1. N,2,3-Trimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo[1,2-

a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide (46). The

title compound was obtained from carboxylic acid 45 (650 mg,

1.87 mmol) and methylamine (2 N solution in THF, 1.87 ml) as de-

scribed in the general procedure: eluant for column chromatogra-

phy: ethyl acetate/triethylamine = 8:2 (v/v); solvent for washing:

diethyl ether; 0.47 g of a colourless solid (70% yield): mp 286–

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

379

287 °C; ¹H NMR (DMSO-d₆, 200 MHz): d = 2.03 (m_c, 1H), 2.20, 2.24,

2.35 (s, m_c, s, 9H), 2.79 (d, 3H), 2.99 (m_c, 4H), 7.36 (m_c, 4H), 7.90 (s,

1H), 8.34 (q, 1H).

2.33, 2.38 (s, m_c, 4H), 2.64–3.14 (m, 4H), 3.30 (br s), 3.50 (m_c,

2H), 7.31 (m_c, 4H), 7.83 (s, 1H); HRMS (ESI) m/z C₂₅H₂₈N₃O₂

[M+H]⁺Calcd: 402.2176. Found: 402.2175.

4.1.16.2. N-Cyclopropyl-2,3-dimethyl-2⁰,3⁰,7,8-tetrahydrospiro-

[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carbox-

amide (47). The title compound was obtained from carboxylic acid

45 (650 mg, 1.87 mmol) and cyclopropylamine (0.26 ml) as de-

scribed in the general procedure: eluant for column chromatogra-

phy: ethyl acetate/triethylamine = 8:2 (v/v); solvent for

crystallization: ethyl acetate, addition of diethyl ether; 0.49 g of a

colourless solid (68% yield): mp 196–197 °C; ¹H NMR (DMSO-d₆,

200 MHz): d = 0.60 (m_c, 2H), 0.72 (m_c, 2H), 2.04 (m_c, 1H), 2.15–

2.40, 2.20, 2.35 (m, 2s, 9H), 2.82–3.10 (m, 5H), 7.32 (m_c, 4H),

7.85 (s, 1H), 8.43 (d, 1H); HRMS (ESI) m/z C₂₄H₂₆N₃O₂[M+H]⁺

Calcd: 388.2020. Found: 388.2016.

4.1.16.7. 2,3-Dimethyl-6-(piperidin-1-ylcarbonyl)-2⁰,3⁰,7,8-tet-

rahydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]

(52). The title compound was obtained from carboxylic acid 45

(600 mg, 1.72 mmol) and piperidine (0.34 ml) as described in the

general procedure: eluant for column chromatography: ethyl ace-

tate/triethylamine = 8:2 (v/v); solvent for crystallization: ethyl

acetate; 0.56 g of a slightly pink solid (78% yield), further puriﬁca-

tion by preparative HPLC: mp 159–160 °C; ¹H NMR (DMSO-d₆,

200 MHz): d = 1.53 (br s, 6H), 2.06 (m_c, 1H), 2.20, 2.23 (s, m_c,

5H), 2.33 (s, m_c, 4H), 2.65–3.10 (m, 4H), 3.30 (br s), 3.60 (br s,

2H), 7.34 (m_c, 4H), 7.75 (s, 1H); HRMS (ESI) m/z C₂₆H₃₀N₃O₂

[M+H]⁺Calcd: 416.2333. Found: 416.2331.

4.1.16.3. 6-(Azetidin-1-ylcarbonyl)-2,3-dimethyl-2⁰,3⁰,7,8-tetra-

hydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene] (48).

The title compound was obtained from carboxylic acid 45

(600 mg, 1.72 mmol) and azetidine (0.23 ml) as described in the

general procedure: eluant for column chromatography: ethyl ace-

tate/triethylamine = 8:2 (v/v); solvent for crystallization: ethyl

acetate; 0.32 g of a colourless solid (48% yield), further puriﬁca-

tion by preparative HPLC: mp 213–214 °C; ¹H NMR (DMSO-d₆,

200 MHz): d = 2.07 (m_c, 1H), 2.20, 2.35, 2.20–2.35 (2s, m, 11H),

2.97 (m_c, 4H), 4.07 (br s, 4H), 7.33 (m_c, 4H), 7.85 (s, 1H). Anal.

Calcd for C₂₄H₂₅N₃O₂: C, 74.39; H, 6.50; N, 10.84. Found: C,

73.98; H, 6.51; N, 10.74; HRMS (ESI) m/z C₂₄H₂₆N₃O₂[M+H]⁺

Calcd: 388.2020. Found: 388.2013.

4.1.16.8. 2,3-Dimethyl-6-(morpholin-1-ylcarbonyl)-2⁰,3⁰,7,8-tet-

rahydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]

(53). The title compound was obtained from carboxylic acid 45

(600 mg, 1.72 mmol) and morpholine (0.30 ml) as described in

the general procedure: eluant for column chromatography: ethyl

acetate/triethylamine = 8:2 (v/v); solvent for washing: diethyl

ether; 0.58 g of a colourless solid (81% yield): mp 223–224 °C; ¹H

NMR (DMSO-d₆, 200 MHz): d = 2.06 (m_c, 1H), 2.20, 2.23 (s, m_c,

5H), 2.33 (s, m_c, 4H), 2.65–3.10 (m, 4H), 3.35 (br s, 2H), 3.66 (br

s, 6H), 7.38 (m_c, 4H), 7.81 (s, 1H). Anal. Calcd for C₂₅H₂₇N₃O₃: C,

71.92; H, 6.52; N, 10.06. Found: C, 71.84; H, 6.51; N, 9.95;

HRMS (ESI) m/z C₂₅H₂₈N₃O₃[M+H]⁺Calcd: 418.2125. Found:

418.2123.

4.1.16.4. 6-[(3-Fluoroazetidin-1-yl)carbonyl]-2,3-dimethyl-2⁰,3⁰,

7,8-tetrahydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,

1⁰-indene] (49). The title compound was obtained from carboxylic

acid 45 (500 mg, 1.44 mmol) and 3-ﬂuoroazetidine hydrochloride

(0.33 g, 2.96 mmol) as described in the general procedure: eluant

for column chromatography: ethyl acetate/triethylamine = 8:2

(v/v); solvent for crystallization: ethyl acetate/diethyl ether/ace-

tone; 0.43 g of a colourless solid (74% yield): mp 177–178 °C; ¹H

NMR (DMSO-d₆, 200 MHz): d = 2.03 (m_c, 1H), 2.20, 2.25 (s, m_c,

6H), 2.36 (s, 3H), 2.99 (m_c, 4H), 4.18, 4.41 (br s, m_c, 4H), 5.31,

5.60 (2 m_c, 1H), 7.32 (m_c, 4H), 7.90 (s, 1H); HRMS (ESI) m/z

C₂₄H₂₅FN₃O₂[M+H]⁺Calcd: 406.1925. Found: 406.1933.

4.1.16.9. 2,3-Dimethyl-N-phenyl-2⁰,3⁰,7,8-tetrahydrospiro[imi-

dazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide

(54). The title compound was obtained from carboxylic acid 45

(600 mg, 1.72 mmol) and aniline (0.31 ml, 3.40 mmol) as described

in the general procedure: eluant for column chromatography:

ethyl acetate/triethylamine = 8:2 (v/v); solvent for crystallization:

ethyl acetate; 0.32 g of a colourless solid (44% yield); mp 201–

202 °C; ¹H NMR (DMSO-d₆, 200 MHz): d = 2.07 (m_c, 1H), 2.23,

2.25 (s, m_c, 6H), 2.40 (s, 3H), 3.01 (m_c, 4H), 7.12 (m_c, 1H), 7.35

(m_c, 6H), 7.75 (d, 2H), 8.13 (s, 1H), 10.40 (s, 1H); HRMS (ESI) m/z

C₂₇H₂₆N₃O₂[M+H]⁺Calcd: 424.2020. Found: 424.2033.

4.1.16.10. N-Methoxy-N,2,3-Trimethyl-2⁰,3⁰,7,8-tetrahydro-

spiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-car-

boxamide (55). The title compound was obtained from

carboxylic acid 45 (650 mg, 1.87 mmol) and N,O-dimethylhydr-

oxylamine hydrochloride (0.36 g) as described in the general

procedure: eluant for column chromatography: ethyl acetate/tri-

ethylamine = 8:2 (v/v); solvent for washing: diethyl ether; 0.34 g

of a colourless solid (47% yield), further puriﬁcation by prepara-

tive HPLC: mp 140–141 °C; ¹H NMR (DMSO-d₆, 200 MHz):

d = 2.07 (m_c, 1H), 2.21, 2.23 (s, m_c, 5H), 2.34, 2.39 (s, m_c, 4H),

2.66–3.06 (m, 4H), 3.30 (br s), 3.60 (s, 3H), 7.31 (m_c, 4H), 7.91

(s, 1H); HRMS (ESI) m/z C₂₃H₂₆N₃O₃[M+H]⁺Calcd: 392.1969.

Found: 392.1961.

4.1.16.5. 6-[(3,3-Diﬂuoroazetidin-1-yl)carbonyl]-2,3-dimethyl-

2⁰,3⁰,7,8-tetrahydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-

9,1⁰-indene] (50). The title compound was obtained from carbox-

ylic acid 45 (500 mg, 1.44 mmol) and 3,3-diﬂuoroazetidine

hydrochloride (0.37 g, 2.86 mmol) as described in the general

procedure: eluant for column chromatography: ethyl acetate/tri-

ethylamine = 8:2 (v/v); solvent for crystallization: ethyl acetate;

0.30 g of a colourless solid (49% yield): mp 174–175 °C; ¹H NMR

(DMSO-d₆, 200 MHz): d = 2.05 (m_c, 1H), 2.20, 2.23 (s, m_c, 6H),

2.37 (s, 3H), 3.00 (m_c, 4H), 4.57 (br s, 4H), 7.32 (m_c, 4H), 7.99 (s,

1H); HRMS (ESI) m/z C₂₄H₂₄F₂N₃O₂[M+H]⁺Calcd: 424.1831.

Found: 424.1841.

4.1.16.6. 2,3-Dimethyl-6-(pyrrolidin-1-ylcarbonyl)-2⁰,3⁰,7,8-tet-

rahydrospiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]

(51). The title compound was obtained from carboxylic acid 45

(600 mg, 1.72 mmol) and pyrrolidine (0.28 ml) as described in

the general procedure: eluant for column chromatography: ethyl

acetate/triethylamine = 8:2 (v/v); solvent for washing: diethyl

ether; 0.38 g of a colourless solid (55% yield), further puriﬁcation

by preparative HPLC: mp 215–216 °C; ¹H NMR (DMSO-d₆,

200 MHz): d = 1.88 (m_c, 4H), 2.05 (m_c, 1H), 2.20, 2.22 (s, m_c, 5H),

4.1.17. Ethyl 8-{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}-7-

[(2E)-2-(5-ﬂuoro-2,3-dihydro-1H-inden-1-ylidene)ethyl]-2,3-

dimethylimidazo[1,2-a]pyridine-6-carboxylate (56)

In a ﬂask ﬁlled with argon, 5-ﬂuoro-1-methyleneindane 24

(4.10 g, 27.7 mmol) and catalyst 16 (440 mg, 0.70 mmol) was

added to a solution of 40 (5.80 g, mixture with 41, 71 wt%,

9.9 mmol) in dry dichloromethane (30 ml). The reaction mixture

was stirred for 1 day at 40 °C and concentrated. Puriﬁcation by col-

umn chromatography [150 g of silica gel, eluant: petrol ether, then

380

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

petrol ether/ethyl acetate = 9:1 (v/v)] afforded a mixture of the title

compound with untransformed starting material 40. The mixture

was suspended in ethyl acetate and the undissolved solid isolated

by ﬁltration. The mother liquor was concentrated, triturated with

diethyl ether, and the resulting solid isolated by ﬁltration. The

two crops were combined [2.1 g, mixture of the title compound

(85 wt%) with starting material 40 (15 wt%), 34% corrected yield]:

¹H NMR (DMSO-d₆, 200 MHz): d = 0.37 (s, 6H), 0.92, 0.99 (d, s,

12H), 1.23 (t, 3H), 1.83 (m_c, 1H), 2.31, 2.39 (2s, 6H), 2.72 (m_c,

2H), 2.95 (m_c, 2H), 3.83 (m_c, 2H), 4.25 (q, 2H), 5.77 (m_c, 1H), 6.94

(m_c, 1H), 7.06 (m_c, 1H), 7.33 (m_c, 1H), 8.25 (s, 1H); HRMS

(ESI) m/z C₃₁H₄₂FN₂O₃Si [M+H]⁺Calcd: 537.2943. Found:

537.2928.

3H), 2.22, 2.28, 2.29, 2.39 (s, m_c, 2s, 13H), 2.90, 3.17 (2 m_c, 4H), 4.34

(q, 2H), 7.19 (m_c, 3H), 8.29 (s, 1H).

4.1.21. 5⁰-Fluoro-2,3-dimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imi-

dazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxylic

acid (60)

A suspension of crude 58 (780 mg, 1.98 mmol) in ethanol

(7 ml) and 6 N potassium hydroxide solution (1.8 ml) was stirred

for 1.5 h at 60 °C. The reaction mixture was quenched with ice-

water (100 ml) and a pH value of 5 was adjusted by addition of

4 N hydrochloric acid solution. The aqueous solution was ex-

tracted with dichloromethane (3ꢁ 50 ml). The combined organic

phases were dried over magnesium sulfate and concentrated un-

der reduced pressure. The solid residue was suspended in ethyl

acetate. The title compound was isolated by ﬁltration (200 mg

4.1.18. Ethyl 8-{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}-2,3-

dimethyl-7-[(2E)-2-(4-methyl-2,3-dihydro-1H-inden-1-ylidene)-

ethyl]imidazo[1,2-a]pyridine-6-carboxylate (57)

of

a

colourless solid, 28% yield): mp 292–293 °C; ¹H NMR

(DMSO-d₆, 200 MHz): d = 2.09 (m_c, 1H), 2.22, 2.25, 2.38 (s, m_c, s,

9H), 2.99, 3.23 (2 m_c, 4H), 7.14 (m_c, 2H), 7.40 (dd, 1H), 8.30 (s,

1H); HRMS (ESI) m/z C₂₁H₂₀FN₂O₃[M+H]⁺Calcd: 367.1452. Found:

367.1442.

In a ﬂask ﬁlled with argon, catalyst 16 (170 mg, 0.27 mmol)

was added in several portions to a solution of 40 (2.20 g, mix-

ture with 41, 90 wt%, 4.8 mmol) and 4-methyl-1-methylenein-

dane 25 (1.52 g, 10.5 mmol) in dry dichloromethane (11 ml).

The reaction mixture was stirred for 17 h at 40 °C and concen-

trated. Puriﬁcation by column chromatography [ﬁrst column:

150 g of silica gel, eluant: petrol ether, then petrol ether/ethyl

acetate = 9:1 (v/v); second column: 100 g of silica gel, eluant:

petrol ether/ethyl acetate = 16:1 (v/v)] afforded a mixture of

the title compound (50 mol%) with untransformed starting

material (50 mol%). This mixture (1.95 g, 43% corrected yield)

was used as starting material for the synthesis of 59: ¹H NMR

(DMSO-d₆, 200 MHz): d = 0.37 (s, 6H), 0.93, 1.00 (d, s, 12H),

1.25 (t, 3H), 1.83 (m_c, 1H), 2.19, 2.31, 2.40 (3 s, 9H), 2.73 (m_c,

2H), 2.82 (m_c, 2H), 3.87 (m_c, 2H), 4.27 (q, 2H), 5.80 (m_c, 1H),

7.04 (m_c, 3H), 8.25 (s, 1H); HRMS (ESI) m/z C₃₂H₄₅N₂O₃Si

[M+H]⁺Calcd: 533.3194. Found: 533.3201.

4.1.22. 2,3,4⁰-Trimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo[1,2-

a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxylic acid (61)

A suspension of 59 (340 mg, 0.87 mmol) in ethanol (3 ml)

and 6 N potassium hydroxide solution (0.8 ml) was stirred for

2 h at 60 °C. A pH value of 5 was adjusted by addition of

4 N hydrochloric acid solution. Dichloromethane (50 ml) and

water (50 ml) was added and the phases were separated. The

aqueous phase was extracted with dichloromethane (2ꢁ

50 ml). The combined organic phases were dried over magne-

sium sulfate and concentrated under reduced pressure. The so-

lid residue was puriﬁed by column chromatography [50 g of

silica gel, eluant: dichloromethane/methanol = 15:1 (v/v)]. Evap-

oration of the corresponding fractions afforded the title com-

pound (310 mg of

a yellowish solid, 98% yield): mp 218–

219 °C; ¹H NMR (DMSO-d₆, 200 MHz): d = 2.08 (m_c, 1H), 2.21,

2.22, 2.29, 2.37 (s, m_c, 2s, 12H), 2.90, 3.19 (2 m_c, 4H), 7.19

(m_c, 3H), 8.27 (s, 1H).

4.1.19. Ethyl 5⁰-ﬂuoro-2,3-dimethyl-2⁰,3⁰,7,8-tetrahydrospiro-

[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxy-

late (58)

4.1.23. 5⁰-Fluoro-N,N,2,3-tetramethyl-2⁰,3⁰,7,8-tetrahydro-

spiro[imidazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-

carboxamide (62)

A suspension of 56 (2.10 g, 85 wt%, 3.32 mmol) in 85% phospho-

ric acid (20 ml) was heated at 50 °C (pre-heated oil bath). After a

period of 30 min, a yellow solution was obtained. Stirring was con-

tinued for 2 h. The reaction mixture was poured into ice-water

(100 ml) and a pH value of 8–9 was adjusted by addition of 10 N

sodium hydroxide solution. The aqueous solution was extracted

with dichloromethane (3ꢁ 50 ml). The combined organic phases

were dried over magnesium sulfate and the solvent was evapo-

rated in vacuo. The crude product (0.80 g of a green foam) was

used as starting material for the synthesis of 60: ¹H NMR (CDCl₃,

200 MHz): d = 1.36 (t, 3H), 2.10 (m_c, 1H), 2.22, 2.25, 2.39 (s, m_c, s,

9H), 3.00, 3.14 (2 m_c, 4H), 4.34 (q, 2H), 7.15 (m_c, 2H), 7.40 (dd,

1H), 8.30 (s, 1H).

The title compound was obtained from carboxylic acid 60

(200 mg, 0.55 mmol) and dimethylamine (2 N solution in THF,

0.55 ml, 1.1 mmol) as described in the general procedure (cf. Sec-

tion 4.1.16): eluant for column chromatography: ethyl acetate/tri-

ethylamine = 8:2 (v/v); solvent for washing: diethyl ether; 80 mg

of

a

colourless solid (38% yield): mp 231–232 °C; ¹H NMR

(DMSO-d₆, 200 MHz): d = 2.08 (m_c, 1H), 2.21, 2.26, 2.33 (s, m_c, s,

9 H), 2.71 (m_c, 2H), 2.91, 2.98, 3.03 (s, m_c, s, 8H), 7.15 (m_c, 2H),

7.42 (dd, 1H), 7.78 (s, 1H); HRMS (ESI) m/z C₂₃H₂₅FN₃O₂[M+H]⁺

Calcd: 394.1925. Found: 394.1919.

4.1.20. Ethyl 2,3,4⁰-trimethyl-2⁰,3⁰,7,8-tetrahydrospiro[imidazo-

[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxylate (59)

A suspension of 57 (1.90 g, 65 wt%, 2.3 mmol) in 85% phospho-

ric acid (20 ml) was heated for 2 h at 50 °C (pre-heated oil bath).

The reaction mixture was poured into ice-water (100 ml) and a

pH value of 8–9 was adjusted by addition of 10 N sodium hydrox-

ide solution. The aqueous solution was extracted with dichloro-

methane (3ꢁ 50 ml). The combined organic phases were dried

over magnesium sulfate and the solvent was evaporated in vacuo.

The crude product was puriﬁed by column chromatography [100 g

of silica gel, eluant: petrol ether/ethyl acetate = 1:1 to 6:4 (v/v)].

This afforded the pure title compound (340 mg of a colourless so-

lid, 38% corrected yield]: ¹H NMR (DMSO-d₆, 200 MHz): d = 1.36 (t,

4.1.24. N,N,2,3,4⁰-Pentamethyl-2⁰,3⁰,7,8-tetrahydrospiro[imi-

dazo[1,2-a]pyrano[2,3-c]pyridine-9,1⁰-indene]-6-carboxamide

(63)

The title compound was obtained from carboxylic acid 61

(300 mg, 0.83 mmol) and dimethylamine (2 N solution in THF,

0.83 ml, 1.66 mmol) as described in the general procedure (cf. Sec-

tion 4.1.16): eluant for column chromatography: ethyl acetate/tri-

ethylamine = 8:2 (v/v); solvent for washing: diethyl ether; 260 mg

of a colourless solid (80% yield): mp 234–235 °C; ¹H NMR (DMSO-

d₆, 200 MHz): d = 2.05 (m_c, 1H), 2.20, 2.25, 2.29, 2.33 (s, m_c, 2s,

12H), 2.70 (m_c, 2H), 2.90, 2.92, 3.03 (m_c, 2s, 8H), 7.20 (m_c, 3H),

7.77 (s, 1H); HRMS (ESI) m/z C₂₄H₂₈N₃O₂[M+H]⁺Calcd:

390.2176. Found: 390.2182.

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

381

4.1.25. 2-Methyleneindane (65)

1H), 4.82 (d, 1H), 5.01 (m_c, 1H), 5.06 (m_c, 1H), 6.00 (ddt, 1H),

7.23 (m_c, 3H), 7.40 (m_c, 1H).

4.1.25.1. Synthesis of 65 from 1,3-dihydro-2H-inden-2-one. At a

temperature of ꢀ15 to 0 °C (acetone/dry ice), a solution of 1,3-

dihydro-2H-inden-2-one 64 (1.00 g, 7.6 mmol) in THF (15 ml)

was added over a period of 20 min to a mixture of titanium tetra-

chloride (1.70 ml, 15.5 mmol) and magnesium turnings (1.48 g,

60.9 mmol) in dichloromethane (22 ml). The reaction mixture

was stirred for 0.75 h at 0 °C and for 0.5 h at room temperature.

The precipitate was removed by ﬁltration and washed with dichlo-

romethane (50 ml). At a temperature of 0 °C, the ﬁltrate was trea-

ted with saturated potassium carbonate solution (80 ml) and the

resulting suspension ﬁltered over Celite. The Celite pad was

washed with dichloromethane (50 ml). The aqueous phase was ex-

tracted with dichloromethane (2ꢁ 20 ml). The combined organic

phases were dried over sodium sulfate, ﬁltered and concentrated

in vacuo. The residue (870 mg of a yellow oil, ¹H NMR analysis:

approximately 22% of 65) was puriﬁed by column chromatography

(70 g of silica gel, eluant: hexane) and the title compound isolated

in 15% yield (150 mg): ¹H NMR (DMSO-d₆, 200 MHz): d = 3.64 (m_c,

4H), 5.08 (m_c, 2H), 7.19 (m_c, 4H).

4.1.28. (2-Allylphenyl)methanol (69)

A

solution of 2-[(2-allylbenzyl)oxy]tetrahydro-2H-pyran 68

(40.0 g, 172 mmol) in 1 N hydrochloric acid (240 ml) and methanol

(240 ml) was stirred for 20 h at 50 °C. The reaction mixture was ex-

tracted with diethyl ether (4 ꢁ 200 ml). The combined organic

phases were dried over sodium sulfate and evaporated to dryness.

The crude product (27.6 g of a yellow oil) was puriﬁed by column

chromatography [800 g of silica gel, eluant: hexane/ethyl ace-

tate = 3:2 (v/v)]. This afforded 16.3 g of the title compound (64%

yield): ¹H NMR (CDCl₃, 200 MHz): d = 3.47 (d, 2H), 4.70 (s, 2H),

5.00 (m_c, 1H), 5.08 (m_c, 1H), 6.00 (ddt, 1H), 7.26 (m_c, 3H), 7.40

(m_c, 1H).

4.1.29. 1-Allyl-2-(chloromethyl)benzene (70)

Over a period of 30 min, thionyl chloride (17.5 ml, 28.7 g, dis-

solved in 50 ml of dichloromethane) was added to a solution of

(2-allylphenyl)methanol 69 (16.0 g, 108 mmol) in dichlorometh-

ane (200 ml). The reaction mixture was stirred for 2 h at room tem-

perature, cooled to 5 °C, and diluted with ice-water (200 ml). A pH

value of 8 was adjusted by addition of 6 N sodium hydroxide solu-

tion (120 ml) and saturated sodium bicarbonate solution (50 ml).

The phases were separated and the aqueous phase was extracted

with dichloromethane (2ꢁ 100 ml). The combined organic phases

were dried over sodium sulfate and concentrated under reduced

pressure. The crude product (17.9 g of a yellow oil) was dissolved

in pentane (50 ml) and aluminium oxide (20 g) added. The suspen-

sion was stirred for 20 min and ﬁltered over a bed of aluminium

oxide (30 g). The ﬁlter cake was washed with 200 ml portions of

pentane. The combined ﬁltrates were evaporated to dryness. The

title compound was obtained in 89% yield (15.9 g of a colourless

oil): ¹H NMR (CDCl₃, 200 MHz): d = 3.52 (d, 2H), 4.60 (s, 2H), 5.00

(m_c, 1H), 5.25 (m_c, 1H), 5.98 (ddt, 1H), 7.27 (m_c, 4H).

4.1.25.2. Synthesis of 65 by intramolecular Heck reaction of 70.

Tetrakis(triphenylphosphine)palladium (5.40 g, 4.7 mmol) was

added to

a solution of 1-allyl-2-(chloromethyl)benzene 70

(15.5 g, 93 mmol) in acetonitrile (150 ml) and triethylamine

(19.7 ml, 14.3 g, 141 mmol). The reaction mixture was heated for

70 min at 85 °C and ﬁltered. The ﬁlter cake was washed with ace-

tonitrile (100 ml). The combined ﬁltrates were evaporated in the

presence of silica gel (20 g). The residue was placed on top of a col-

umn ﬁlled with silica gel (600 g) and the title compound eluted

with pentane. Evaporation of the corresponding fractions afforded

the title compound (9.14 g of a colourless oil, 75% yield): ¹H NMR

(DMSO-d₆, 200 MHz): d = 3.63 (m_c, 4H), 5.08 (m_c, 2H), 7.19 (m_c,

4H).

4.1.26. 2-[(2-Bromobenzyl)oxy]tetrahydro-2H-pyran (67)

3,4-Dihydro-2H-pyran (29.3 ml, 27.2 g, 323 mmol) and Amber-

lyst 15 (33 g) was added to a suspension of (2-bromophenyl)meth-

anol 66 (50.0 g, 267 mmol) in n-hexane (250 ml) and diethyl ether

(26 ml). The solution was stirred for 45 min at room temperature

and added on top of a column ﬁlled with 400 g of silica gel and

33 g of Amberlyst 15. The title compound (51.5 g of a colourless

oil, 71% yield) was eluted with a mixture of n-hexane and diethyl

ether [10:1 (v/v)]: ¹H NMR (CDCl₃, 200 MHz): d = 1.51–1.96 (m,

6H), 3.56 (m_c, 1H), 3.93 (m_c, 1H), 4.56 (d, 1H), 4.77 (t, 1H), 4.83

(d, 1H), 7.15 (m_c, 1H), 7.32 (m_c, 1H), 7.54 (m_c, 2H).

4.1.30. 7-[2-(1,3-Dihydro-2H-inden-2-ylidene)ethyl]-6-

(dimethylcarbamoyl)-2,3-dimethyl-imidazo[1,2-a]pyridin-8-yl

pivalate (71)

4.1.30.1. Synthesis of 71 by cross-metathesis in the presence of

2nd generation Grubbs catalyst 15. In a ﬂask ﬁlled with argon, 2-

methyleneindane 65 (583 mg, 4.48 mmol) and catalyst 15 (80 mg,

0.09 mmol) was added to a solution of 28 (0.40 g, 1.1 mmol) in dry

1,2-dichloroethane (20 ml). The reaction mixture was stirred for

2 h at 80 °C and concentrated. Puriﬁcation by column chromatog-

raphy (20 g of silica gel, eluant: ethyl acetate) afforded a mixture

of the title compound and starting material 28 in a 1:1 ratio

(400 mg of a green oil). This mixture was dissolved in dry 1,2-

dichloroethane (20 ml) and treated with 2-methyleneindane 65

(583 mg, 4.48 mmol) and catalyst 15 (80 mg, 0.09 mmol). After a

reaction time of 2 h at 80 °C, the solvent was evaporated and the

residue (1 g of a brown solid) puriﬁed by column chromatography

(20 g of silica gel, eluant: ethyl acetate). Evaporation of the corre-

sponding fractions afforded the following products: 410 mg of

1,1⁰,3,3⁰-tetrahydro-2,2⁰-biindene 72 in the form of a foamy solid;

233 mg of the title compound (44% yield) in the form of beige crys-

tals and 190 mg of a mixture of the title compound with untrans-

formed starting material 28 (1:2-ratio) in the form of a green oil:

analytical data cf. Sections 4.1.30.2 and 4.1.31.

4.1.27. 2-[(2-Allylbenzyl)oxy]tetrahydro-2H-pyran (68)

In a ﬂask ﬁlled with argon, 2-[(2-bromobenzyl)oxy]tetrahydro-

2H-pyran 67 (50.0 g, 184 mmol, dissolved in 80 ml of dry THF) was

added slowly to a suspension of magnesium turnings (5.0 g,

206 mmol) in dry THF (50 ml). The Grignard reaction was started

by addition of iodine and by gentle heating. The reaction tempera-

ture was maintained between 40 and 45 °C. After complete addi-

tion of 67 (45 min), stirring was continued for 15 min and the

reaction mixture was heated to reﬂux for 1.5 h. At a temperature

of 30–35 °C, a solution of allyl bromide (21.6 ml, 30.2 g, 245 mmol)

in THF (50 ml) was added over a period of 30 min. The reaction

mixture was stirred for 17 h at room temperature and quenched

at 10 °C (ice-cooling) by addition of saturated ammonium chloride

solution (200 ml). The phases were separated and the aqueous

phase was extracted with ethyl acetate (4ꢁ 200 ml). The combined

organic phases were dried over sodium sulfate and evaporated to

dryness. This afforded 40.8 g of the title compound (colourless

oil, 95% yield): ¹H NMR (CDCl₃, 200 MHz): d = 1.49–1.94 (m_c, 6H),

3.46 (d, 2H), 3.56 (m_c, 1H), 3.92 (m_c, 1H), 4.51 (d, 1H), 4.71 (t,

4.1.30.2. Synthesis of 71 by cross-metathesis in the presence of

Hoveyda–Grubbs catalyst 16. In a ﬂask ﬁlled with argon, a solu-

tion of 28 (8.0 g, 23.3 mmol) in 1,2-dichloroethane (90 ml) was

heated at 80 °C. With the help of a dosing pump, a solution of 2-

methyleneindane 65 (15.2 g, 116.7 mmol) in 1,2-dichloroethane

(40 ml) was added over a period of 4 h. Portions of catalyst 16

382

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

(4ꢁ 300 mg, 1.92 mmol) were added 0, 1, 2 and 3 h after start of

the reaction. After complete addition of 2-methyleneindane 65,

the solvent was evaporated in vacuo. The crude product was puri-

ﬁed by column chromatography [800 g of silica gel, eluant: petrol

ether, then petrol ether/ethyl acetate = 1:9 (v/v)]. Evaporation of

the corresponding fractions afforded the title compound in 27%

yield (2.85 g of a green foam): ¹H NMR (DMSO-d₆, 200 MHz):

d = 1.39 (s, 9H), 2.27, 2.37 (2s, 6H), 2.78, 2.79 (2s, 6H), 3.31 (br s),

3.62 (m_c, 4H), 5.29 (m_c, 1H), 7.23 (m_c, 4H), 8.10 (s, 1H).

dium hydroxide solution. The phases were separated and the

aqueous phase was extracted with dichloromethane (2ꢁ 10 ml).

The combined organic phases were dried over sodium sulfate

and concentrated in vacuo. The crude product was puriﬁed by col-

umn chromatography [silica gel, eluant: dichloromethane/metha-

nol/ammonia (25% aqueous solution) = 20:1:0.1 (v/v/v)]. The title

compound was isolated in 17% yield (62 mg of a colourless solid)

and puriﬁed further by preparative HPLC: mp 237–238 °C; ¹H

NMR (DMSO-d₆, 200 MHz): d = 2.15, 2.18 (m_c, s, 5H), 2.31 (s, 3H),

2.65 (m_c, 2H), 2.91 (s, 3H), 3.03 (s, 3H), 3.17 (s, 4H), 7.22 (m_c,

4H), 7.75 (s, 1H). The reactions described in Table 2, entries 4

and 5, were performed under similar conditions (modiﬁcations:

addition of the substrate to 85% phosphoric acid at room tempera-

ture, direct neutralization of the reaction mixture by addition of

sodium bicarbonate solution).

4.1.31. 1,1⁰,3,3⁰-Tetrahydro-2,2⁰-biindene (72)

A solution of 2-methyleneindane 65 (200 mg, 1.54 mmol) and

2nd generation Grubbs catalyst 15 (34 mg, 0.04 mmol) in 1,2-

dichloroethane (2 ml) was heated to 80 °C for 2 h and the solvent

was evaporated. The residue was puriﬁed by column chromatogra-

phy (silica gel, eluant: n-pentane). The title compound was isolated

in 78% yield (140 mg of

a

grey solid): ¹H NMR (DMSO-d₆,

4.1.33.3. Synthesis of 74 by cyclisation in the presence of

hydrobromic acid (Table 2, entry 7). A mixture of 73 (100 mg,

0.27 mmol) and hexadecyltributylphosphonium bromide (15 mg,

200 MHz): d = 3.63 (s, 8H), 7.24 (m_c, 8H).

4.1.32. 7-[2-(1,3-Dihydro-2H-inden-2-ylidene)ethyl]-8-hydroxy-

N,N,2,3-tetramethylimidazo[1,2-a]pyridine-6-carboxamide (73)

A solution of lithium hydroxide (173 mg, 4.1 mmol) in water

(1.4 ml) was added to a solution of 71 (650 mg, 1.4 mmol) in 1,4-

dioxane (6.3 ml). The reaction mixture was heated at 100 °C for

1 h and poured on a mixture of ice (20 g), saturated ammonium

chloride solution (70 ml) and dichloromethane (70 ml). The phases

were separated and the aqueous phase was extracted with dichlo-

romethane (2ꢁ 10 ml). The combined organic phases were dried

over sodium sulfate and concentrated under reduced pressure.

The crude product (450 mg of a brown oil) was puriﬁed by column

chromatography [100 g of silica gel, eluant: dichloromethane/

methanol = 20:1 (v/v)]. The title compound was isolated as green

oil (408 mg, 80% yield). Further puriﬁcation of the title compound

(280 mg) was achieved by crystallization from ethyl acetate/

diethyl ether [1:10 (v/v)]: After a period of 16 h, a colourless crys-

talline solid (205 mg) was isolated by ﬁltration: mp 217–218 °C;

¹H NMR (DMSO-d₆, 200 MHz): d = 2.31, 2.33 (2s, 6H), 2.76 (s,

3H), 2.85 (s, 3H), 3.36 (d, 2H), 3.59, 3.67 (2 br s, 4H), 5.40 (m_c,

1H), 7.21 (m_c, 4H), 7.64 (s, 1H).

0.03 mmol) in hydrobromic acid (150 ll, 48% aqueous solution)

was heated for 2 h at 110 °C. The reaction mixture was poured

on dichloromethane and saturated sodium bicarbonate solution.

The phases were separated and the aqueous phase was extracted

with dichloromethane (2ꢁ). The combined organic phases were

dried over sodium sulfate and the solvent was evaporated. The

crude product was puriﬁed by column chromatography [30 g of sil-

ica gel, eluant: dichloromethane/methanol/ammonia (25% aqueous

solution) = 20:1:0.1 (v/v/v)] and 11 mg of the impure title com-

pound were isolated (11% yield): analytical data cf. Sections

4.1.33.2 and 4.1.33.6.

4.1.33.4. Synthesis of 74 by cyclisation in the presence of

ﬂuorosulfuric acid (Table 2, entry 8). At a temperature of

ꢀ75 °C, ﬂuorosulfuric acid (150

ll) was added dropwise to a solu-

tion of 73 (100 mg, 0.27 mmol) in nitropropane (1 ml). After a per-

iod of 30 min, the reaction mixture was poured on ice-water

(10 ml) and dichloromethane (10 ml). The phases were separated

and the aqueous phase was extracted with dichloromethane (2ꢁ

5 ml). The combined organic phases were dried over sodium sul-

fate and the solvent was evaporated in vacuo. The brown residue

was puriﬁed by column chromatography (20 g of silica gel, eluant:

dichloromethane/methanol). The title compound (8 mg, 8% yield)

was isolated in the form of a brown oil: analytical data cf. Sections

4.1.33.2 and 4.1.33.6.

4.1.33. N,N,2,3-Tetramethyl-1⁰,3⁰,7,8-tetrahydrospiro[imidazo-

[1,2-a]pyrano[2,3-c]pyridine-9,2⁰-indene]-6-carboxamide (74)

4.1.33.1. Synthesis of 74 by cyclisation in sulfuric acid/acetic

acid (Table 2, entry 3). A solution of 73 (50 mg, 0.13 mmol) in ace-

tic acid (0.4 ml) was heated to 80 °C and sulfuric acid (0.2 ml) was

added dropwise. After a period of 10 min, the reaction mixture was

poured on ice (5 g), saturated sodium bicarbonate solution (20 ml)

and dichloromethane (15 ml). The phases were separated and the

aqueous phase was extracted with dichloromethane (2ꢁ 5 ml).

The combined organic phases were dried over sodium sulfate

and the solvent was evaporated. The crude product (33 mg of a yel-

low oil) was puriﬁed by column chromatography [15 g of silica gel,

eluant: dichloromethane/methanol/ammonia (25% aqueous solu-

tion) = 20:1:0.1 (v/v/v)] and the title compound was isolated in

16% yield (8 mg of a colourless solid containing minor impurities).

The reaction described in Table 2, entry 2 was conducted under

analogous conditions except for the fact that sulfuric acid was

added at 10 °C (before heating the reaction mixture to 50 °C): ana-

lytical data cf. Sections 4.1.33.2 and 4.1.33.6.

4.1.33.5. Synthesis of 74 by cyclisation in the presence of

tetrakis(2,2,2-triﬂuoroacetoxy)stannane (Table 2, entry 9).

A

solution of 73 (100 mg, 0.27 mmol) and tetrakis(2,2,2-triﬂuoro-

acetoxy)stannane (203 mg, 0.28 mmol) in nitromethane (3 ml)

was heated for 0.75 h at 120 °C and poured on a mixture of ice

(10 g), saturated sodium bicarbonate solution (20 ml) and dichlo-

romethane (20 ml). The phases were separated and the aqueous

phase was extracted with dichloromethane (2ꢁ 5 ml). The com-

bined organic phases were dried over sodium sulfate and con-

centrated. The brown solid residue (105 mg) was puriﬁed by

column chromatography [15 g of silica gel, eluant: dichloro-

methane/methanol/ammonia (25% aqueous solution) = 20:1:0.1

(v/v/v)] and the title compound was isolated in 14% yield

(14 mg of a yellow oil): analytical data cf. Sections 4.1.33.2

and 4.1.33.6.

4.1.33.2. Synthesis of 74 by cyclisation in 85% phosphoric acid

(Table 2, entry 6). At a temperature of 130 °C, 73 (370 mg,

0.98 mmol) was dissolved portionwise in phosphoric acid

(85 wt% in water). The reaction mixture was stirred for 15 min at

130 °C and poured into a mixture of ice (50 g) and dichlorometh-

ane (50 ml). A pH value of 7 was adjusted by addition of 10 N so-

4.1.33.6. Synthesis of 74 by cyclisation in the presence of boron

triﬂuoride diethyl etherate (Table 2, entry 11). A solution of 73

(800 mg, 2.13 mmol) and boron triﬂuoride diethyl etherate

(5.28 ml, 5.97 g, 42.0 mmol) in 1,1,2,2-tetrachloroethane (30 ml)

was heated at 80 °C for 16 h. The reaction mixture was diluted with

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

383

sodium bicarbonate solution and dichloromethane. The phases

were separated. The organic phase was dried over sodium sulfate

and the solvent was evaporated in vacuo. The residue was puriﬁed

by column chromatography [150 g of silica gel, eluant: ethyl ace-

tate/methanol = 10:1 (v/v)]. Evaporation of the corresponding frac-

tions afforded 354 mg of a yellow oil. The title compound (304 mg

of a colourless solid, 39% yield) was obtained by crystallization

speciﬁc activity) and 1 mM Na–ATP (high grade), reaction vol-

ume: 101 l. Preparation of malachite green reagent: 2 parts of

l

malachite green stock solution (1.2 M in H₂O, protected from

light and used within 12 weeks) were mixed with 1 part of

ammoniumheptamolybdate tetrahydrate stock solution (42 g/l

in 4 N HCl) and kept for 30 min at room temperature prior to

use. A pipes/tris buffer based solution with sucrose and MgCl₂

was prepared. Nigericin and enzyme were added to reach the ﬁ-

nal concentrations mentioned above. Eighty microlitres per well

of this mixture were placed into 96-well ﬂat-bottomed plates

(clear, polystyrol, Greiner bio-one). Ten microlitres per well of

KCl (1 mM ﬁnal) was used for stimulation of the H⁺/K⁺-ATPase

activity. Test substances were dissolved as 10 mM solutions in

100% DMSO. One microlitre of substance solution was added in

dilutions ranging from 1 ꢁ 10^ꢀ4to 1 ꢁ 10^ꢀ9M (ﬁnal). The enzy-

from

a mixture of ethyl acetate (0.5 ml) and diethyl ether

(10 ml): mp 240–241 °C; ¹H NMR (DMSO-d₆, 400 MHz): d = 2.15,

2.18 (m_c, s, 5H), 2.31 (s, 3H), 2.65 (m_c, 2H), 2.91 (s, 3H), 3.03 (s,

3H), 3.16 (s, 4H), 7.21 (m_c, 4H), 7.75 (s, 1H); HRMS (ESI) m/z

C₂₃H₂₆N₃O₂[M+H]⁺Calcd: 376.2020. Found: 376.1993. The reac-

tion described in Table 2, entry 10 was conducted under analogous

conditions.

4.1.34. 8-Hydroxy-7-[2-(1H-inden-2-yl)ethyl]-N,N,2,3-tetra-

methylimidazo[1,2-a]pyridine-6-carboxamide (75)

matic reaction was started by addition of 10

The assay was incubated for 30 min at room temperature. The

reaction was stopped by addition of 150 l of malachite green

ll ATP (1 mM ﬁnal).

A solution of 73 (80 mg, 0.21 mmol) in formic acid (0.8 ml)

was heated under microwave irradiation for 30 min at 150 °C.

The reaction mixture was poured on ice-water (30 ml) and

dichloromethane (20 ml) and a pH value of 7.5 was adjusted

by addition of concentrated aqueous ammonia solution. The

phases were separated and the aqueous phase was extracted

with dichloromethane (2ꢁ 10 ml). The combined organic phases

were dried over sodium sulfate and the solvent was evaporated.

The residue was puriﬁed by column chromatography [30 g of sil-

ica gel, eluant: dichloromethane/methanol = 50:1 (v/v)] and

14 mg of the title compound were isolated (18% yield): ¹H

NMR (DMSO-d₆, 400 MHz): d = 2.32 (s, 3H), 2.35 (s, 3H), 2.65

(m_c, 2H), 2.82, 2.83 (s, br s, 5H), 3.00 (s, 3H), 3.35 (s, 2H), 6.52

(s, 1H), 7.08 (m_c, 1H), 7.17 (m_c, 1H), 7.24 (m_c, 1H), 7.37 (m_c,

1H), 7.67 (s, 1H).

l

reagent and incubated for another 15 min prior to photometric

reading of the plate at 680 nm in a PowerWave HT Microplate

spectral photometer (BioTek). The results were analyzed with

GraphPad Prism software (Version 4.02) to calculate IC₅₀values

by sigmoidal curve ﬁtting. ‘Enzyme’ refers to H⁺/K⁺-ATPase-con-

taining vesicles prepared from hog gastric mucosa as described

in Rabon, E. C.; Im, W. B.; Sachs, G. Methods Enzymol. 1988,

157, 649–654.

4.2.2. ¹⁴C-Dimethylaminopyridine accumulation in intact

gastric glands

Gastric acid secretion is stimulated by gastrin, histamine and

acetylcholine via the receptors on the parietal or the enterochro-

mafﬁn-like cell. These physiologic stimuli inﬂuence the intracellu-

lar cyclic AMP and Ca²⁺levels, thus leading to relocation and

activation of H⁺/K⁺-ATPase. Instead of the physiologic agonists,

the membrane-permeant dibutyryl-cyclic AMP was used to stim-

ulate receptor-independent acid secretion in isolated gastric

glands. Accumulation of the weak base ¹⁴C-dimethylaminopyri-

dine (¹⁴C-AP) in the acidic compartment of the canaliculi serves

as an indirect measure of acid secretion and forms the basis of

measurement of acid secretion in this in vitro model of the mam-

malian stomach. Intact gastric glands were prepared from anes-

thesized New Zealand rabbits (weight 2–3 kg) by high-pressure

perfusion of the stomach, separation of the fundic mucosa and

subsequent collagenase digestion of fragments of the mucosa

(Berglindh, T; Helander, H. F.; Obrink, K. J. Acta Physiol. Scand.

1976, 97(4), 401–414; Berglindh, T.; Obrink, K. J. Acta Physiol.

Scand. 1976, 96(2), 150–159). After the gastric glands were

washed several times, they were suspended in Krebs–Henseleit

solution containing 2 mg/ml rabbit serum albumin and 2 mg/ml

glucose. Glands were incubated for 30 min at 37 °C in a shaker

4.1.35. 7-{(2E)-4-[2-(Chloromethyl)phenyl]but-2-en-1-yl}-8-

{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}-N,N,2,3-

tetramethylimidazo[1,2-a]pyridine-6-carboxamide (76)

In a ﬂask ﬁlled with argon, 1-allyl-2-(chloromethyl)benzene 70

(200 mg, 1.20 mmol) and Hoveyda–Grubbs catalyst 16 (30 mg,

0.05 mmol) was added to a solution of 29 (250 mg, 0.60 mmol) in

dry dichloromethane (5 ml). The reaction mixture was stirred for

20 h at 45 °C and concentrated. Puriﬁcation by column chromatog-

raphy [50 g of silica gel, eluant: dichloromethane/metha-

nol = 100:1 (v/v)] afforded the title compound in 72% yield

(240 mg of a yellow oil containing impurities): ¹H NMR (DMSO-

d₆, 400 MHz): d = 0.35 (s, 6H), 0.95 (s, 6H), 1.05 (d, 6H), 1.80 (m_c,

1H), 2.28 (s, 3H), 2.30 (s, 3H), 2.69 (s, 3H), 2.91 (s, 3H), 3.40 (m_c,

4H), 4.71 (s, 2H), 5.45 (m_c, 2H), 7.24 (m_c, 3H), 7.38 (m_c, 1H), 7.76

(s, 1H).

4.2. Biochemistry

bath (200 osc/min) in the presence of 0.125

(113 Ci/ mol) at pH 7.4. Glands were stimulated with 1 mM

dibutyryl cAMP in absence or presence of the corresponding

inhibitor (concentration range 3 nM–100 M). The reaction was

lM

¹⁴C-AP

4.2.1. Determination of inhibitory activity in a competitive

binding assay against H⁺/K⁺-ATPase from hog gastric mucosa

Given data are the mean IC₅₀values from 2 to 3 independent

determinations. The malachite green assay modiﬁed from Yoda

and Hokin (Biochem. Biophys. Res. Commun. 1970, 40, 880–886)

was used for the determination of H⁺/K⁺-ATPase IC₅₀: Lanzetta,

P. A.; Alvarez, L. J.; Reinach, P. S.; Candia, O. A. Anal. Biochem.

1979, 100, 95–97. Pipes [piperazine-1,4-bis(2-ethanesulfonic

acid)], sucrose, nigericin, Na–ATP and malachite green were pur-

chased from Sigma–Aldrich, tris [tris(hydroxymethyl)amino-

methane], KCl and ammoniumheptamolybdate tetrahydrate

from Merck, and MgCl₂from Fluka. Final assay concentrations:

4 mM Pipes/8 mM tris buffer, pH 7.4, 0.25 M sucrose, 1 mM

l

stopped by centrifugation (10 s at 20,000g). After centrifugation,

the accumulation of ¹⁴C-AP in the glands was calculated as fol-

lows: radioactivity was measured in an aliquot of the supernatant

(200 ll) and in the precipitate after dissolution in 1 ml of 1 N

NaOH. In order to calculate the amount of protein, the Eppendorf

tubes were weighed empty, with protein (wet weight) and with

freeze–dried protein (dry weight). This ratio of supernatant and

pellet protein radioactivity was used to calculate the accumula-

tion of ¹⁴C-AP in the glands. The inhibitor concentration required

to achieve 50% inhibition (IC₅₀) of ¹⁴C-AP accumulation was

determined by ﬁtting the equation for the expected inhibition

pattern to the data points.

KCl, 1 mM MgCl₂, 0.5–1

lg/100 ll nigericin (1:1 ratio with en-

zyme), 0.5–1 g/100

l

l enzyme (dependent on K⁺-stimulated,

384

A. M. Palmer et al. / Bioorg. Med. Chem. 17 (2009) 368–384

5. Design of novel P-CABs, Part V: Palmer, A. M.; Grobbel, B.; Jecke, C.; Brehm, C.;

4.3. Physical chemistry

Zimmermann, P. J.; Buhr, W.; Feth, M. P.; Simon, W. A.; Kromer, W. J. Med.

Chem. 2007, 50, 6240.

6. Modlin, I. M.; Sachs, G. Acid Related Diseases: Biology and Treatment; Schnetztor-

Verlag: Konstanz, 1998.

7. Heartburn, hiatal hernia, and gastroesophageal reﬂux disease (GERD); NIH

Publication No. 03-0882, June 2003; http://digestive.niddk.nih.gov/ddiseases/

pubs/gerd/index.htm.

8. H. pylori and peptic ulcer; NIH Publication No. 05-4225, October 2004; http://

digestive.niddk.nih.gov/ddiseases/pubs/hpylori/index.htm.

9. Parsons, M. E.; Keeling, D. J. Expert Opin. Investig. Drugs 2005, 14, 411.

10. Fass, R.; Shapiro, M.; Dekel, R.; Sewell, J. Aliment. Pharmacol. Ther. 2005, 22,

79.

4.3.1. General

The determination of dissociation constants (pK_a) and lipophil-

icity [logP, logD (pH 7.4)] were performed on a Sirius GL pK_aana-

lyzer speciﬁcally designed for pH-metric pK_aand 1-octanol/water

partition coefﬁcient measurements (Sirius Analytical Instruments

Ltd, Forest Row, UK).

4.3.2. Determination of dissociation constants

11. Vakil, N. Aliment. Pharmacol. Ther. 2004, 19, 1041.

12. Andersson, K.; Carlsson, E. Pharmacol. Ther. 2005, 108, 294.

13. Kaminski, J. J.; Bristol, J. A.; Puchalski, C.; Lovey, R. G.; Elliott, A. J.; Guzik, H.;

Solomon, D. M.; Conn, D. J.; Domalski, M. S.; Wong, S.-C.; Gold, E. H.; Long, J. F.;

Chiu, P. J. S.; Steinberg, M.; McPhail, A. T. J. Med. Chem. 1985, 28, 876.

14. Kaminski, J. J.; Hilbert, J. M.; Pramanik, B. N.; Solomon, D. M.; Conn, D. J.; Rizvi,

R. K.; Elliott, A. J.; Guzik, H.; Lovey, R. G.; Domalski, M. S.; Wong, S.-C.;

Puchalski, C.; Gold, E. H.; Long, J. F.; Chiu, P. J. S.; McPhail, A. T. J. Med. Chem.

1987, 30, 2031.

The pK_avalues of the investigated compounds were determined

by potentiometric co-solvent titrations in 0.15 mol/l KCl solutions

in the range of pH 2.0 to pH 11.0 at 25 °C using methanol as co-sol-

vent in varying portions and 0.5 mol/l KOH and HCl as titrants,

respectively. Linear extrapolation to 0% co-solvent-content was

performed by the Yasuda–Shedlovsky plot method implemented

in the software ReﬁnementPro 2 from SIRIUS (Avdeef, A.; Box, K.

J.; Comer, J. E. A., Gilges, M.; Hadley, M.; Hibbert, C.; Patterson,

W.; Tam, K. Y. J. Pharm. Biomed. Anal. 1999, 20, 631–641).

15. Kaminski, J. J.; Puchalski, C.; Solomon, D. M.; Rizvi, R. K.; Conn, D. J.; Elliott, A. J.;

Lovey, R. G.; Guzik, H.; Chiu, P. J. S.; Long, J. F.; McPhail, A. T. J. Med. Chem. 1989,

32, 1686.

16. Folded conformation: phenyl ring directed toward and over the imidazo[1,2-

a]pyridine ring system; extended conformation: phenyl ring oriented out and

away from the heterocyclic nucleus.

17. Gold, E. H.; Kaminski, J. J.; Puchalski, D. US 4468400.

18. Kaminski, J. J.; Wallmark, B.; Briving, C.; Andersson, B. M. J. Med. Chem. 1991,

34, 533.

19. Palmer, A. M.; Nettekoven, U. Tetrahedron: Asymmetry 2007, 18, 2381.

20. Palmer, A. M.; Nedden, H.; Zanotti-Gerosa, A. Tetrahedron: Asymmetry 2008, 19,

1310.

21. Palmer, A.; Zimmermann, P. J.; Buhr, W.; Brehm, C.; Senn-Bilﬁnger, J.; Simon,

W. A.; Postius, S.; Kromer, W. International Patent Application, WO

2008071766.

22. Van de Weghe, P.; Eustache, J.; Cossy, J. Curr. Top. Med. Chem. 2005, 5, 1461.

23. Vernall, A. J.; Abell, A. D. Aldrichim. Acta 2003, 36, 93.

24. Connon, S. J.; Blechert, S. Angew. Chem. Int. Ed. 2003, 42, 1900.

25. For further details regarding catalyst 15 see Grubbs, R. H. Tetrahedron 2004, 60,

7117.

26. For further details regarding catalyst 16 see Hoveyda, A. H.; Gillingham, D. G.;

Van Veldhuizen, J. J.; Kataoka, O.; Garber, S. B.; Kingsbury, J. S.; Harrity, J. P. A.

Org. Biomol. Chem. 2004, 2, 8.

4.3.3. Determination of distribution coefﬁcients

The distribution coefﬁcients between 1-octanol and aqueous

KCl solution were determined at 25 °C by potentiometric titrations

in the range of pH 2.0 to pH 11.0. The titrations were performed in

mixtures of 0.15 mol/l KCl solution and water saturated 1-octanol

with varying 1-octanol portions using 0.5 mol/l KOH and HCl as ti-

trants, respectively. The logD values in dependence of pH were ob-

tained by least squares ﬁtting of the experimental data to a

theoretical function of the distribution coefﬁcient D (Reﬁnement-

Pro2): Comer, J.; Tam, K.; Lipophilicity proﬁles: Theory and Measure-

ment, in Pharmakokinetic Optimization in Drug Research: Biological,

Physicochemical and Computational Strategies. Testa, B., van de

Waterbeemd, H., Folkers, G., Guy, R., Eds.; VHCA: Zurich, 2001;

pp. 275–304.

27. For further details regarding catalyst 17 see Wakamatsu, H.; Blechert, S. Angew.

Chem. 2002, 114, 2509.

28. For further details regarding catalyst 18 see Grela, K.; Harutyunyan, S.;

Michrowska, A. Angew. Chem. 2002, 114, 4210.

Acknowledgments

We are grateful to Ms. C. Jecke, Mr. B. Grobbel, Ms. M. David, Ms.

I Hartig-Beecken and Ms. B. Bössenecker for their technical assis-

tance. We would also like to thank Prof. K. Grela (Polish Academy

of Sciences, Warsaw) for general discussions about cross-metathe-

sis and for the provision with catalysts 17 and 18.

29. For further details regarding catalyst 19 see Fürstner, A.; Guth, O.; Düffels, A.;

Seidel, G.; Liebl, M.; Gabor, B.; Mynott, R. Chem. Eur. J. 2001, 7, 4811.

30. Ohta, T.; Ikegami, H.; Miyake, T.; Takaya, H. J. Organomet. Chem. 1995, 502,

169.

31. Justik, M.; Koser, G. Molecules 2005, 10, 217.

32. TBTU = O-(1H-benzotriazol-1-yl)-N,N,N⁰,N⁰-tetramethyluronium

tetraﬂuoroborate, see, for example, Montalbetti, C. A. G. N.; Falque, V.

Tetrahedron 2005, 61, 10827.

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Spiro(imidazo[1,2-a]pyrano[2,3-c]pyridine-9-indenes) as inhibitors of gastric acid secretion

DOI: 10.1016/j.bmc.2008.10.055

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