Structure-activity relationships involving the catechol subunit of rolipram

Liioorganic & kfedicinal Chemistly Letters, Vol. 4, NO. 15. pp. 1855-1860, 1994 Copyright0 1994 E1swie.r Science L.td Printed in Great Britain. All rights ~su~ed~ G940-894X/94 $7.00+0.00

0960-894X(94)00247-9

Structure-Activity

Relationships Involving the Catechol Subunit of Rolipram

Jeffrey A. Stafford,**= Paul L. Feldman,= Brian E. Marron,= Frank J. Schoenen,a Nicole L. Valvano,a Rayomand J. UnwaBa,b Paul L. Domanico,e E. Sloan Brawley , CMichael A. Leesnitzer,e Dudley A. Rose,e and Robert W. Doughertyd Glaxo Research Institute 5 Moore Drive Research Triangle Park, NC 27709 a Department of Medicinal Chemistry b Department of Molecular Sciences c Department of Biochemistry d Department of Cellular Biochemistry

Abstract: Structure-activity rolipram (l), arediscussed. Rolipram CAMP-specific for chronic

involving the aromatic ring of the selective PDE IV inhibitor,

(1) is a selective inhibitor of

[(R, S)-4-(3-cyclopentoxy-4-methoxyphenyl)-2-pyrrolidinone] phosphodiesterase

inflammatory

diseases

medicinal

chemistry

synthesis

of PDE IV inhibitors

pyrrolidinone

relationships

type IV (PDE IV).l Due to the wide interest in developing through

selective

effort aimed at improving

ring.2 Less attention,

Letter we report structure-activity

derived

inhibition

however,

agents

of PDE IV, there has been a considerable

the potency of rolipram. A number of groups have reported the

from rolipram,

relationships

therapeutic

in which

the modifications

has been directed to modifications of rolipram derivatives

were

made to the

of the catechol ring.3 In this

in which the aromatic ring has been

modified.

Rolipram (1)

2 (n=l) 3 (n=2)

4

The exact nature in which rolipram binds to PDE IV is unclear due to a lack of structural data on the enzyme. Our primary goal therefore was to prepare derivatives

of the catechol that would help us ascertain the

nature in which the catechol ethers interact with the enzyme. The methylenedioxyderivatives,

24 and 3,s respectively,

were prepared and found to be considerably

IV than rolipram itself (see Table). A similar loss in potency was observed acetal4.6 1855

and ethylenedioxyphenyl

less potent inhibitors

of PDE

with the derived cyclohexylidene

1856

J. A. STAFFORDef al.

We reasoned that the loss in activity observed with compounds 2-4 perhaps stemmed from an improper orientation of the electron lone pairs associated with the oxygen atoms of the catechol. If one or both of these oxygens are involved in hydrogen bonding to an amino acid residue on the enzyme surface, clearly then the spatial geometry of these electron lone pairs is critical.7 The cyclic framework holding the catechol subunit of 2-4 forces the general dipoles created by the lone pairs to be directed away from each other (Figure la). However, it is possible that the optimum binding mode for rolipram and related derivatives is such that these dipoles are generally oriented in the same direction (Fig lb). Indeed, the minimum energy conformation of rolipram (Fig lc) has the two alkyl groups of the catechol ethers oriented similarly to the depiction in Figure

Figure lb

Figure la

Figure lc

In order to test this hypothesis we required aromatic derivatives of rolipram wherein the oxygen lone pairs are constrained to meet the geometric arrangement shown in Figure lb. If, in fact, the orientation in Fig. lb is required one might expect that compounds possessing this arrangement would exhibit equal or improved potency versus rolipram for inhibiting PDE IV. By simply anchoring the methoxy group at the 4-position to the ring we designed the benzofuran 5, dihydrobenzofuran 6, and dihydrobenxopyran 7, Each of these compounds meets the generalized criterion set forth by Fig. lb. At the outset of this work, however, we were not certain of the enzyme’s tolerance for the added substituent on the aromatic group in 5-7. Additionally,

the effect of

lowering the basicity of one of the catechol oxygens when going to a benzofuran (i.e., sp3-hybridization in 1; sp2-hybridization in 5) was unknown.

5

6 (n=l) I (n=2)

The synthesis of benzofuran 5 is shown in Scheme 1. Knoevenagel condensation between 3-furaldehyde (8) and diethyl malonate provided the unsaturated diester 9. DIBAL reduction of 9 (toluene, -78 “C), followed by acetylation of the derived diol afforded 10. Direct formation of the benzofuran ring from 10 was then accomplished

using a palladium-catalyzed

cyclocarbonylation

reaction recently reported by Hidai and

The catechot subunit of rolipram

1857

coworkers.g Thus, treatment of 10 with Pd(PPh3)2ClZ (5 mol %) and triethylamine

in toluene under an

atmosphere of CO (IOOQpsi, 175 “C, 11 hr) provided, after silica gel chromatography, benzofuran IX in 45% yield. Saponification

of the acetate esters (LiUH, H2Um)

selectively ~y~lo~nty~at~

(~y~lu~ntyl

bromide, K$O3,

provided an intermediate

DMF) KUgive the ben~fu~~

dial, which was alcohol 12 in 74%

yield from fl. Oxidation of the benzylie alcohol with MnU2 then delivered aldehyde 13 in 39% yield. With W in hand we followed a previously established three-step sequence to append the pyrrolidinone ring.sa Thus, Homer-Emmons homologation of 13 provided an unsaturated ester, which upon treatment with CH3N@ and tetramethylguanidine (TlMG), afforded an internxxliate nitroester resulting from conjugate addition of CH3NO2. Catalytic reduction of the nitro group afforded an intermediate amino ester, which undergoes spontaneous cyclization to provide the rolipram derivative 5 in an overall yield of 53% from 13.10

Scheme 1

EtO&XH $02Et piperidine, PhCHs

1. Dll3At 2. AC20 -

t , (MeO)~POCH&O&le MnO2 CHC13

LiHMDS, THF 2. CH3N02, TMG, $5 “C 3. Ha, Raney-Ni, CHsUH

cc%I’

The syntheses of mlipram derivatives 6 and 7 began from dihydrobenzofuran 14 and dihydrobenzopyran 15, respectively, and is shuwn beIow in Scheme 2.11 Thus, O-alkylation (cyclopentyl bromide, QCO3, DMF) provided the aldehydes 16 and 17, which were then converted to 6 and 7 by the same three-step sequence described above in the synthesis uf $12

1858

J. A. STAFFORD et al.

Scheme

2

CHO 14 (ml) 15 (n=2)

Test compounds

7 (n=2)

were measured

both for their ability to inhibit the catalytic activity of PDE IV (Ki)‘3 binding site (Q). t4,15 The data are shown in the Table.

and for their ability to bind to the rolipram high-affinity As mentioned Consistent

above, compounds with

our

dihydrobenzopyran

2-4 were considerably activity

hypothesis,

derivative

is restored

less potent inhibitors in the

of PDE IV than (+)-rolipram.

dihydrobenzofuran

derivative

6 and

7, which are similar in their ability to inhibit PDE IV, Ki = 3.69 and 1.17 pM,

respectively. Perhaps most noteworthy

is the benzofuran

analog 5, whose PDE IV inhibitory

activity (Ki = 0.26 pM)

is nearly equal to rolipram. It appears that lowering the basicity of the ether oxygen does not necessarily deleterious

effect on enzyme inhibition.

reasonable

to suggest that the in-plane sp2-hybridized

accept a hydrogen

Within the framework

bond than its counterpart,

6.l6 This being the case, the enhanced due to the greater flexibility

of our hydrogen-bonding

lone pair of the benzofuran

out-of-plane

sp3-hybridized

activity of the dihydrobenzopyran

of a six-membered

hypothesis,

have a

it is then

oxygen is better oriented to

lone pair in the dihydrobenzofuran 7, relative to 6, is easily understood

ring versus a five-membered

ring. This added flexibility

in 7

allows the oxygen to position itself more. favorably for hydrogen bonding. Finally, the rolipram binding data for this set of compounds

parallel the enzyme inhibition

approximately

lower than its Ki, compounds

lo-fold

data. Indeed, like rolipram itself, which has a Q that is 5 and 7 display significantly

greater binding affinity than

PDE IV inhibition. Table PDE IV Inhibition (K;. UM)

(f)-Rolipram

Rolipram Binding (Kd. p.h4)

0.19

0.01

2

> loo

> loo

3

> loo

> loo

4

63.9

16.5

5

0.26

0.03

6

3.69

4.00

7

1.17

0.21

1859

The catcchol subunit of rohpram

In summary, we have presented the PDE IV inhibitory activity of a number of rolipram derivatives, which were designed to explore the nature in which the catechol ether moiety interacts with the enzyme. Our goal was to understand the optimum spatial arrangement of the catechol ethers, not simply to assess the steric tolerance at each oxygen. We have provided evidence that the spatial ~angem~nt

of the oxygen lone pairs of

the catechol ethers am critically important with respect to the PDE IV inhibitory properties of rolipram and its derivatives. The ability to design potent inhibitors of PDE IV, which are based on rolipram, should be facilitated by the present study. Acknowledgement The authors thank the ICOS Corporation (Bothell, WA) for supplying the PDE IV that was used in the enzyme experiments. References and Notes 1. Schwabe, U.; Miyake, M.; Ohga, Y.; Daly, 3. Mol. Patrol.

1976,900.

2. (a) Koc, B. K.; Lebel, L. A.; Nielsen, J. A.; Russo, L. L.; Saccamano, N. A.; Vinick, F. J.; Williams, I. H. Drug Dev. Res. 1990,21,

135. (b) Pinto, I. L.; Buckle, D. R.; Readshaw, S. A.; Smith, D. G. Bioorg. Med.

Chem. Lett. 1993,3, 1743. (c) Backstrom, R.; Honkanen, E.; Raasmaja, A.; Linden, I.-B. Int. Pat. Appl. (PCT)

WG 91/16303 (26 Apr 1991). (d) Bagli, J. F.; Lomb&o,

L. J.; Skotnicki, J. S. gut. Par. Appl. 0 511 865 Al (30

Apr 1992). (e) Bender, P. E.; Christensen, S. B. Znt. Pat. Appl. (PCT) WO 93/07141(2 Ott 1992). 3. (a) Marivet, M. C.; Bourguignon, J. J.; Lugnier, C.; Mann, A.; Stoclet, J.-C.; Wermuth, C.-G. J. Med. Chem. 1989,32,1450.

(b) Huth, A.; Beetz, I.; Schumann, I. Tefra~dr~~ 1989,45,6679.

4. This com~und

was prepared adding from piperonal using an es~b~sh~ synthetic route.3aTh~ data for 2 are as follows: m.p. 153-55 OC. 1H NMR (CDCl3,300 MHz): 6 2.44 (dd, 1, J = 8.8, 16.8). 2.70 (dd, 1, J = 8.8, 16.8), 3.36 (dd, 1, J = 2.0,7.3), 3.62 (m. l), 3.72 (m, 1). 5.94 (d, 2,J = 2.7), 6.41 (bs, I), 6.75 (m, 3). Anal calcd

for CtlHllN03:

C, 64.38; H, 5.40, N, 6.83. Found: C, 64.23; H, 5.39; N, 6.74.

5. This compound was prepared beginning from 1,4-benzodioxan-6-carboxaldehyde using an established synthetic route.3a The data for 3 are as follows: m.p. 98-99 OC. 1H NMR (CDCl3, 300 MHz): 6 2.43 (dd, 1, J = 9.0, 16.8). 2.67 (dd, 1, J = 8.8, 16.8), 3.35 (dd, 1, J= 7.8,9.3), 3.58 (m, l), 3.72 (dd, 1, J = 8.3,9.3), 4.24 (s, 4), 6.71 (m, 2), 6.75 (d, 1, J = l-7), 6.82 (d, 1, J = 8.3). Anal calcd for CrzHl3N03: C, 65.74; H, 5.98; N, 6.39. Found: C!,65.75; H, 6.02; N, 6.40. 6. 4: m.p. 164-65 “C. ‘II NMR (CDCl3, 300 MHz): 8 1.49-1.85 (m, IO), 2.43 (dd, 1, J = 8.9, 17.1), 2.67 (dd, 1, J = 8.9, 16.9), 3.35 (t, 1, J = 8.4). 3.59 (m, 1), 3.72 (t, 1, J = 8.4). 6.65 (m, 3), 6.47 (bs, 1). Anal calcd for C&llgNO3:

C, 70.31; H, 7.01; N, 5.12. Found: C, 70.28; H, 7.06; N, 5.07.

7. We have examined the effect of removing either the methoxy or the cyclopentoxy group, respectively, These two rolipram analogs inhibit PDE IV with a Kt = 26 @I and >lOO PM, respectively, demonstrating the requirement that both ether groups be present for significant PDE IV inhibition. 8. This minimum-energy

rolipram conformation was calculated by semi-empirical (PM3) calculations within

SPARTAN ~avefunction

Inc) and is in good agreement with the recently pubfished crystal structure of (+)-l-

(4-bromobenzyl)-4-(3-(cyclopentoxy)-4-~~ox~henyl)p~oli~n-2-one:

Baures, P. W.; Eggleston, D. S.;

Erhard, K. F.; Cieslinski, L. B.; Torphy, T. J.; Christensen, S. B. J. Med. Cfzem. 1993,36,3274.

J. A. STAPFORD et al.

1860

9. Iwasaki, M.; Kobayashi,

K.; Li, J.-P.; Matsuzaka, H.; Ishii, Y.; Hidai, M. J. Org. Chem. 19% 5X1922.

10.5: mp. 169-70 ‘C. 1H NMR (CDCl3,300

MHz): 6 1.64-1.97 (m, 8), 2.54 (dd, 1, J = 8.5, 16.9), 2.78 (dd, 1,

J= 8.7, 17.0), 3.45 (m, 1), 3.73-3.84 (m, 2), 5.02 (m, l), 5.95 (bs, 1), 6.67 (s, l), 6.71 (d, 1, J= 2.2), 7.05 (d, 1, J = 1.2), 7.61 (d, 1, J = 2.0). Anal calcd for C17H19N03: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.43; H, 6.73; N, 4.93. Stafford, J. A.; Valvano, N. L. J. Org. Chem., in PUSS.

11. The syntheses of 14 and 15 are reported elsewhere:

12. 6: m.p. 121-23 OC. 1H NMR (CDC13, 300 MHz): 6 1.54-1.92 (m, 8), 2.45 (dd, 1, J = 8.9, 16.8), 2.70 (dd, 1, J= 8.5, 16.8),3.20(t,2,J=8.8),

3.37 (dd, l,J=7.1,8.8),

3.62(m,

l), 5.75 (bs, I), 6.60 (s, I), 6.72 (s, 1). Anal calcd for Cl7H2lN03:

1),3.75 (m, 1),4.61 (t. 2,J=8.8),4.84(m, C, 71.06; H, 7.37; N, 4.87. Found: C, 71.14;

H, 7.35; N, 4.84. 7: m.p. 142-45 “C. 1H NMR (CDC13, 300 MHz): 6 1.53-2.03 (m, lo), 2.46 (dd, 1, J = 9.0, 17.1), 2.69 (dd, 1, J = 8.7, 16.8), 2.74 (m, 2), 3.37 (dd, 1, J = 7.3,9.0),

3.58 (m, I), 3.74 (m, l), 4.23 (m, 2), 4.73

(m, 1), 5.59 (bs, l), 6.53 (s, l), 6.58 (s, 1). Anal calcd for C17H2lN03*0.13

H20: C, 71.18; H, 7.72; N, 4.61.

Found: C, 71.16; H, 7.67; N, 4.35. 13. The Ki for a test compound compound

is dissolved

is evaluated against recombinant

human PDE IV enzyme as follows: The test

and serially diluted in 100% DMSO, and then diluted

stepwise

with Hz0 to 10%

DMSO. A 100 ltL reaction is prepared by mixing 10 p.L of a buffer solution, which contains 20 mM TRIS, 10 mM MgC12, 2 mM EDTA, 2 mM DlT, 0.5% n-octyl glycoside, 5 mg/mL metal-free BSA , pHf = 7.5; 10 @L of the test compound

(10-e to lo-1uM final, 1% final DMSO); 10 ltL of the substrate solution containing

CAMP (300 nC final) and 5.3 l..tM 14C-adenosine

addition of 20 ltL of an enzyme solution containing diluted appropriately reaction

is stopped

batchwise

into reaction

50 l@nL

of 5’-nucleotidase

buffer to give 50% turnover of substrate

by the addition

1 PM 3H-

(3 nC final), and 50 l.tL H20. The reaction is initiated by the and hrPDE IV that has been

in 20 min at 23 ‘C. The PDE

of pH 10.0 buffer. The cyclic nucleotide

and the nucleoside

are then

separated by QAE Sephadex A25 anion exchange resin. The filtrate (100 FL) is mixed with 150 l.tL

of scintillation

cocktail and counted in dual channel mode. The Ki is determined

data to the following equation: I = [(Ymax * [II)& three independent

by fitting the dose-response

+ [I])] + Yniin. Values in the Table represent the mean from

determinations.

14. Torphy, T. J.; Stadel, J. M.; Burman, M.; Cieslinski, L. B.; McLaughlin,

M. M.; White, J. R.; Livi, G. P. J.

Biol. Chem. 1992,267,1798. 15. The ability of test compounds

to compete with [3H]rolipram for binding to recombinant

assessed as follows: hrPDE IV is incubated with [3H]rolipram (IO-9 M final concentration) (1O-11 through 10e4 M) in a total volume of 1.0 mL containing mixing

to adsorb

the ligand/receptor

quantified

concentration/response 16. Computer systems

respectively,

liquid

complex.

The mixture

scintillation

is filtered

counting.

ICsn

is ad&d to 2.5% were

within SPARTAN

(Wavefunction

to mimic 5 and 7, 7-methoxybenzofuran

confirmed

(Received in USA 6 June 1994; accepted 22 June 1994)

determined

and the from

correction.

Inc) at a basis set level (6-31G*) performed (5’) and 7-methoxy-2,3-dihydrobenzofuran

that the dipole vectors are oriented differently.

y=O.O, z=O.43 @t=1.06 D); For 7’ the vector coordinates

with

(W/V)

onto glass fiber filters values

curves (n=2) and converted to Ki values using the Cheng-Prusoff

calculations

modelled

by

and test compounds

(mM): NaCl (lOO), Tris-HCI (25), MgCl2 (lo),

CaCl2 (11, and BSA (0.25%, w/v) pH 7.4. After 60 min on ice, hydroxyapatite radioactivity

human PDE IV was

For 5’ the vector coordinates

are x=0.14, y=-0.79, z~l.18 (~~1.49 D).

on (7’),

are x=0.93,