Indian Journal of Chemistry Vol. 3813, December 1999, pp 1331 -1 337

Synthesis of RGD peptidomimetic analogues of 2,5-diketopiperazinet

N V S Ramakrishna*t, T S More, Y Khandelwal, R G Naik, Bansi Lal, R D Gupte & R V S V Vadlamudi

Research Centre, Hoechst Marion Roussel Ltd., LBS Marg, Mulund (W),

Mumbai 400080, India

Received 6 No vember 1998; accepted 24 March 1999

Synthesi s of 3-(mcthy lacetatc)-6-(N-ben zyloxypropy l amino)-2 , 5~diketopiperazine . 5 and its corresponding RGD analogues 9, 11 ,15 and 16 has been achieved and their platelet aggregat ion IIlhlbltory aclivlty evaluated.

Platelet activation and the resulting aggregation have been shown to be associated with various pathological conditions, including cardiovascu lar and cerebrovascu lar thromboembolic disorders such as unstable angina, myocardial infarction , transient ischemic attack and stroke 1,2. The binding of adhesive proteins, such as fibrinogen to the glycoprotein IIbllll a (GP lib/ ilia) receptor on platelets causes platelets to aggregate,,4. The binding of fibrinogen is mediated in part by the arginine-glycine-aspartic acid (RGD) (Figure 1) recognition sequence, which is common to the ad hesive proteins that bind to GP lib/ ili a receptors. Thus, RGD based naturals (snake venom' s) and synthetic peptides6 and monoclonal antibodies7

.8 raised aga inst GP lib/ lila have been

shown to inhibit platelet aggregation.

It is currently believed that inhibition of fibrinogen binding to platelets by blocking of GP lib/lil a receptors is a rational approach for the development of novel antithrombotic drugs for the treatment of a variety of thrombotic conditions that lead to coronary artery and cerebrovascular diseases9

. It has been hypothesized that potent inhibitors could be prepared by incorporating an element of geometric and structural constraints in the RGD6

. Compounds that possess guanidine and carboxylic acid functional groups separated by a spacer of 13-16 atoms may provide the lead structure. We wish to describe in this

tThis work has been presented at the 10·h IUPAC International Conference on Organic Synthesis, held at Banga laore in December 1994. "Current address: Zyd us Research Ccntn:. Cad ida Ilcaltheare Lt d, Zydus Tower. Satellit e Cross Road. Sarkhej-Gandh inagar Il ighway, Ahmedabad 380 0 15.

RGD

A = 13- 16 atoms

Figure 1

paper the synthesis of RG D peptidom imetic analogues of 2,5-diketopiperazine by variation of G Iy­Asp in Arg-Gly-Asp (Figure 1).

Results and Discussion 3-(Methyl acetate )-6-(N-benzyloxypropylal1l ino)-

2,5-diketopiperazine 5 was the key intermediate in the synthesis of RGD pepetidomimetic an alogues of 3,6-disubstituted 2,5-diketopiperazines (Schemes I-III) . Synthesis of 5 was achieved starting from L-orn ithine (Scheme 1). Z-Protected L-orn ith ine 1 was prepared using the method of CuCO}Cu(OH h complex tO fo llowed by treating with benzy loxycarbonyl chloride . a-Amino group of 1 was protected by I-Boc to yield 2. Dimethyl aspartate was condensed with 2 to afford 3. Boc deprotection or a -amino group of.3 with formi c acid and ani sole gave 4. N-benzy loxy-o rnithylas partate 4 was cyc lized to

1332 INDIAN J CH EM, SEC B, DECEMBER 1999

a

z 0

H~~NH BOC -NH I

H COOC~COOCH3 3

c

(3)

Z 0

H~~NH NH2 I COOCH

HCOOC~ 3 3

e

(4)

Z

I~COOH HN I

NH2

(1)

Z

I~COOH HN I

NH I

(2) BOC

Z 0 I 51(4 HN~ Nrl

HN _ ~3COOCH3 1 I(;, '--./

o

(5)

Reagents: (a) CuC03 .CU(O H)2 . NaOH , C1COOCH2 C6Hl , diethyl ether, lOoC, 2N HCl, HIS; (b) Di-Ierl_butyldicarbonate / dioxane, NaHC03 ,HI 0 , RT; ( c) ClCOOCI

Hl , dioanae, diethyl asparta te, 4-methylmorpholine; (d) HCOOH, anisole, RT; (e) n­BuOH, AcO H, 4-methy lmorpholine, reflux

Scheme I

get the des ired compound 5, lI s in g n-BuOHI AcOH/4-methylmorpholine as reported for the preparation of 2-ketop iperazines ll

.

N-Terminal analogues (Scheme II) were prepared by Z-deprotection of 5 followed by condensation w ith various Zr guanidinoamino ac ids to yie ld 8 . Z2-Guan idinoamino acids 6 were prepared from the corresponding amino acids and di-carbobenzoxy-S­Illethyl!sothiourea us ing the reported II procedure. Guan idine-esters 9 were prepared by deprotection of 8 lI s ing CFJSO, H/CF1COOH and the products were isolated as TF A sa lts. G uanidine-acids 11 were prepared from 8 by alka line hydro lys is fo ll owed by Z­c1 eprotection and the products were iso lated as TF A sal ts.

C-Terminal analogues (Scheme III) were prepared by a lkaline hydrolys is of 5 fo ll owed by condensation with various amino esters. Compounds 13 on Z­deprotect ion using H2/Pd-C in MeO H yie lded 14. Amino esters 14 were converted to the corresponding gua nidino ester-TFA salts 15 with di-carbobenzoxy-S­methy l isoth iourea using the reported 12 procedure, foll owed by Z-deprotection of guanid ine group w ith CF:.SO, H/CF,COQl--1. G uanidino-acids 16 were prepared by alka line hydro lysis of 15.

The structu res of the compounds syn thesised were ass igned on the basis of e lementa l ana lyses, IR and 1 H NMR spectra l data (Table I). All th e compounds were tested in human PRP for pla telet aggregat ion in hibitory acti v ity but none of th e m showed

( J

RAMAKRI SHNA et al. : SYNTH ES IS OF RGD ANALOGUES OF 2,5-DlKETOPIPERAZIN E 1333

ZHN r N-Z

SoMe Z I H 0

HN 'y-" N, )l Et3N, CH3CN

( CH3)3SiCI

II R OH

Z / N (6)

Z 0 l/'-... /'--. Jl H2/Pd-C, AcOH

HN '-/ "I -NH

o

HN~.NH HN~COOCH3 MeOH 2 HN~eooeH3

o (5)

o (7)

CICOOEt, 10 ° e , DMF

Scheme II

interesting activ ity at 1.0 ~. However, w hen tested at higher concentrations, fe w compound s showed activity around 30 ~tM concentration, which was above the des ired cut-off for further pharmaco logical evaluation. Though in most of the eva luated compounds, the distance between guanidine group and carboxy l group is 13-16 atoms, no apprec iable inhibitory activ ity could be found .

Experimental Section

General. Melting po ints were determined on a

Bri sto line apparatus and are uncorrected . IR spectra were recorded on a Perkin- Elmer mode l 782 spectrometer. I H N M R spectra were recorded on a Brucker AC P 300 MH z spectrometer with tetramethy ls il ane as interna l standard . Mass spectra were recorded on a Kratos MS 80 mass spectrometer. Co lumn chromatography was performed over sili ca ge l (60-1 20 mesh) purchased from Acme Chemica l Company, Bombay. T LC was carri ed out on Merck s ili ca ge l precoated (0 . 1 mm ) plates and spots were detected e ither with 254 nm UV lamp or Dragendorffs reagent.

1334 I DIA 1 CHEM, SEC B, DECEMBER 1999

o

HN~NH 0 1N NaOH/MeOH

I HN , ~ Jl

o

HN -~'NH 0

I HN , ~ Jl Z I( "-./ -OCH3

o (5)

Z I( "-./ -OH

o (12)

CICOOEt j ,----, H2N- R OMe

O,--!N - Me, 1 0 °c l

H/ Pd-C/MeOH --

j 1)

2)

NH-Z

Z-HN JlS-Me

Et3N, CH3CN

CF3S03H, TFA

Scheme III

Synthesis of 3,6-disubstituted-2,S-diketopiperazine

Z-Protection of L-ornithine (1). I.-Orn ithine (2 0 g, 152 Inmols) was taken in water (30 mL) and heated to boi lin g. and CuCO\Cu(O Il ):> co mpl ex (14 g, 63 mmoles) was added to the reac ti on mixture over a period of 15 min . The reaction mixture was then a llowed to come to room temperatu re over a period of I hr and filtered into a ro und bottom flask. To thi s o lu tion 2N NaOI-l (130 I11L) and benzy loxyca rbonyl

chloride ( 13.64 Ill L 8 mmo les) in ether (30 I11 L) was added and stirred at 20 "c fo r 40 min : aga in a second lot of reagent (Cbz-CI. 13.6-1 mL ) in eth er (70 I1l L) and NaOI-l (30 lllL) was added and the mixture further stirred for 3 hr.

The so lid materi a l thus obtained was liltered and d i so lved in 2N HCI. Through thi s so lution hydrogen sulfid e gas was passed fo r 2 hL Ali er th e reaction was com pleted, it was filtered by add ing superce l. To the mot her liquor. pyridine \Va added till IJII was 6.0. The prec ip itate obta ined was fi Iteree! ane! th e crude

purified by crystalli ati on using hot water, yield 25.00 g (62 %); 11-1 MR (DMSO, 0) 7.56(s, 51-1 , Ar) , 5.04(s, 21-1, C I-I :> Ph), 3.92(1, 21-1. C I-I2 1-1 ), 3. 17(1, 11-1 , C I-I COO I-I), 1.8 -1 .9(m, 4 1-1 , CH1).

Boc-protection of N-bcnzoyloxyclu'bonyl-L-o .... ithine (2). Z-Pro tccted ornithine (5 g, 19 Illlllo les) was taken in dry dioxa ne. Nal-lC01 in water was add ed to it . To thi s reaction mi xtu re di-(C/"(-butyldicarbonate in dioxane was added dropwi se over

a period of 15 min . The reacti o"n mi xture was further stirred for I hI' at room temperature. After compl et ion of the reacti on. dioxane was removed, and th e res idue d iluted with water and extracted with ether . The aq ueo us layer was cooled, acidi fied with citric acid. and th en extracted with ethy l aceta te. The combined et hyl acetate ex tract was washed with brine, dri ed ovcr Na ~ SOI and the so lvent evaporated under vac uulll . y icld 6.2 g (91 % ); 11-1 1R (DMSO. 0): - .<t(s. 51 L I\r ), 5.0(s. 21 L C l-bPh ), 3.92( 1. 211. C II ~I /I ), 3. 17( 1. Ill, CI/COO I/ ). 1.9- 1. 8( 111 , -I I L C II ~ ) . 1.52(s, 91-1. I-bu ty l).

RAMAKRISHNA el al. : SYNTHESIS OF RGD ANALOGUES OF 2,5-D1KETOPIPERAZrNE 1335

Compd R mp (OC)

17S-79

175-77

ISc -(CH 2k 15S-59

Table I--Characteri sation data o f compounds 9, II , 15 and 16

mol. formula

C2oH27N60gSFJ

IHNMR

(DMSO-d6'o 7.40-7 .SS(m, 4H, Ar), 4.4(s. 2H , C H2-N H),4.25 (t, I H, C H), 4.0(t, I H, C H), 3.6(s, 3H, OCH), 3.25 (t, 2H. CH2-NH ),2.7(d, 2H. CH2-CO), 1.9- I.S (m, 4H, CH2)

7.40-7.S5( m, 4H , Ar), 4.25(t, I H, C H), 4 .0(t, I H. CH), 3.6(s, 3H, OCH3)' 3.25( t, 2H, CH2-

H). 2.7(d. 2H. CHrCO). 1.9- I.S(m. 4H, C 1I 2)

Clg H23 N60gS F.l 7 45 -7 .S5(m, 4H, Ar). 4.2(1, I H, C H), 4 .0(t, 11-1 , C H), 3.25(t, 2H , C H2-N H), 2.7(d, 2H , CH 2-CO), 1.9-I .S(m, 4H. CH2)

CI?H 2,N60~SFJ 7.40-7.S5(m, 4H , Ar). 4.4(s, 2H, Cl-lr NH), 4 .25(1, I H, C H), 4.0(t, I H, C H), 3.25(1.2 11 , C H2-N II ). 2.7(d. 2H , CHT

CO). 1.9- 1.8( m, 4 H, C 112)

C2o ll27N60~SFJ 7.40-7 .85(m, 4 H, Ar) , 4.4(s, 2H, C 112-Nil ). 4.25( 1. I H, CII ), 4.0(1. I H, C II ). 3.6(s, 311. OC II ) , 3.25(1, 211. CHr NI-I) , 2.7(d , 2H . C HrCO). 1.9-1. 8(m. II. CH2)

C1911 2,N60 sS F) 7.40-7.85(m. 4 11 , Ar) . 4.25(1, 11-1 , C II ). 4 .0( t. I H. C II ), 3.6(s. 311 , OCII J). 3.25( 1, m , C I-12-NI-I). 2.7(d . 2H. C 1-I 2-

CO), 1.9- 1.8( m, 4H , C 1I2)

CI7 1-1 29N60 gSr3 4.3(1. III. CII ). 4. 1 (I. III. C I-I). 3.65(s. 3H, OC II 3). 3.4(t. 41-1 , C ll z-NI-I) , 26(d, 411 , C ll rCO). 1.9- 1.8( m, SI-I . C Hz)

C 1?l-I z,N60 gSF) 7.40-7.S5(m, 4 11 , Ar) . 4.4(s, 2H, CI-12-

N il). 4 .25(1. I H, C II ). 4.0(1, I H, C I-I ). 3.25(1. 211. C l-l rN II ). 2 .7(d, 2 1-1 , CHr CO), 1.9-1.8( m, 4 11. CI-12)

CIs l-l 23 N60gSr3 7.45 -7.85(m. 4 11 , Ar). 4 .2(t. 11-1 . C II ), 4.0(1, III , C I-I ). 3.25(t, 2H , C l-l rN H), 2.7(d, 211. C HrCO), 1.9-I. S(m, 4 H, C H2)

Found (%) (Caled) C H N

41.95 4.59 14.57 (42 .25 4.75 14.78)

41.49 (4 1.15

40.26 (40.00

40.32 (4 1.15

41.75 (42.25

41.47 (4 1.15

37.83 (38.20

40.4 1 (4 1.15

40.06 (40.00

4.38 4 .51

14.94 15. 16)

4.47 15.23 4.25 15.55)

4.65 14.66 4.51 15.16)

4.64 14.47 4 .75 14.78)

4.33 14 .74 4.51 15.16)

5112 15.84 5.43 15.73)

4.75 14 .56 4.5 1 15.16)

4.57 15. 13 4.25 15.55)

Preparation of dimethyl N-benzyloxy-N -Boc­ornithylaspartate 3 : 2-t-Boc-amino-5-carbob enzylo­xyaminopentanoi c ac id (2 .8 g, 8 mmoles) was taken 111 dry dioxane and cooled to 10 °C. 4-Methylmorpholine (2. 14 mL, 19 .46 mmo les) and ethy l chloroformate (0.88 mL, 9.20 mmo le) were added and the reaction mi xture was stirred at 10 °C for 30 min , followed by addition of dimethyl aspartate ( 1.23 g, 8 lllmolcs) . T I1 e reacti on mi xture was further stirred for 2 hr at the sa me temperature . After completion of th e reaction, dioxane was removed, and

the res idue diluted with water and then extracted wi th e thyl acetate. Ethy lacetate extract was washed w ith brine, dried over sodium sulphate and concentrated under vac uum, y ield 3 .6 g (92 %); IH NMR (DMSO,

8): 7.45(s, 5H, Ar), 5.0(s, 2 H, C Hr Ph), 3.92(t, 2 H, C I-I 2NI-I ), 3.7(s, 6H , OC I-I 3), 3.40(m, 2 H, C I-I CON I-I), 3 .0(d, 21-1 , CI-I 2CO), 1.9-1 .8(01, 4 H, C H2), 1.48(s, 91-1 , t-buty l).

Prepartion of dimethyl N-benzyloxy-ol'llithylaspartate 4. Dimethyl N-benzyloxy-N~Boc­

o rnithy laspartate (6 g, 12 mmoles) was taken 111

1336 INDIAN J CHEM, SEC B, DECEMBER 1999

anhyd rous formic acid (60 mL), followed by addition of ani sole (2.8 mL, 26 mmoles). The reaction mixture was stirred at room temperature for 2 hr. After com pletion of the reaction, acid was removed under vacuu ll1 . The crude so obta ined was purified over s ilica ge l column using MeOH-CH3CN-CHCb ( I :2:7) as eluent, yield 3.1 3 g (65 %); .IH NMR (DMSO, 0) 7.45(s, 51-1, Ar), 5.0(s, 21-1, CI-I2Ph), 3.92(t, 21-1, C I-I 2N I-I ), 3.9(s, 61-1 , OCI-I3) , 3.45(111 , 21-1 , C I-ICONI-I), 3.0(cl. 2H, CI-I 2CO), 1.9-1.8(m, 41-1, CH2)'

P,'cp:u-ation of 3-(methyl acetate)-6-(N-bcnzyloxypropylamino)-2,5-diketopiper'azine 5. Dimcthy l N-benzy loxy-ornithylaspartate (0.06 g, 0 . .1

ml11ole) was taken in l1-butanol (0 .4 1l11ll0Ies), all L! acetic acid (2 IllL, 0.1 mole) was added followed by the add ition of N-methylmorpholine (0.13 mL. 1.18 minole). The reacti on mi xture was then relluxed for I

hr. After completion of the reaction, solvent was removed, and the crude was purified over silica co lumn eluting with MeOI-l-CI-I3CN-CHCb ( I :2:7), yield 0.055 g (65 %); IH NMR (DM SO, 0) 7.7(s,5H, Ar), 5.45(s, 2H, CI-I2Ph ), 4.7(t, 11-1 , CI-I), 4.35(t, 11-1 , CI-I ), 3.45(t, 21-1, C I-I 2 1-1 ), 3. 15(d, 2H, C I-I 2CO), 1.9-2.2(m, 41-1 , CI-I 2).

Synthesis of N-terminal analogues of 5 General procedure for the pr'eparation of Zr

guanidinoamino acids 6. Corres ponding amino ac id (2 g) was taken in dry acetonitril e, and trimethy lsilyl chloride (1.7 mL, 1.0 eq) added to it dropwise . The reaction mi xture was relluxed at 75 ·C for I hr and then a llowed to attain room temperature. Triethylamine ( 1.0 eq) was then added followed by the addition of Zr.)-met hyli sothiourea (1.0 eq) at room temperature and the mi xture reflux ed overnight. After comp leti on of the reaction excess methanol was added wh ile stirrin g. The solvent was removed , and the crude dissolved in water, ac idif~ ed with 2N HCI , extracted with ethyl acetate, dried over Na2S0.1 and concentrated unde r vacu um . The crud e compo und was purified over silica gel colull1n eluting with MeO H/CHC I.1 gradient, yield 4.25 g (70 %).

Prcparation of 3-(mcthyl acctate)-6-propyl-am ino-2 ,5-dikctopiperazine 7. Compound 5 (1 . 15 g) was taken in dry mcthanol , and 10 % Pd-C (~ 0.2 g) was addcd under nitrogen atmosphcre, fo ll owed by the additi on of acetic acid (- I mL). The reaction mixture was subjected to hydroge nati on at 45 psi 1'0 1' 2 ii I'. After completion of th e reaction, th e cata lyst was filtered off and meth anol concentrated to dryness . The crude was di sso lved in dry ll1ethanol (50 mL), Il ask was protected by calc ium chlor ide gua rd-tube, and to

this solution etheral HCI added till pH 2.0 . Compound 7 separated out slowly. After stirring for I hr, the precipitated so lid 7 was filtered and dried, y ie ld : 0.48 g (75 %).

General procedure for the preparation of 8. Zr guanid inoa lkanoic acid or Z2-guanidinobenzo ic acid (0 .6 g) was taken in dry DMF in a round bottom ed Ilask protected with a calcium chloride guard-tube and cooled to 10 °C. N-Methylmorpholine (0.25 mL) and ethyl chloroformate (0. 17 mL) were added and the reaction mixture was stirred for 30 min at the same temperature foll owed by thc additi on of 3-(methyl acetate )-6-(propylam in o )-2,5 -d ikctopi pera­zine (0.42 g) . The reaction Illi xture was th en stirred overn ight. After complet iOIl or the reaction, th e mixture was diluted wi th water and extracted with ethy l aceta te. The orga llic layer was dried over Na2S01 and conccntrated to dryness . The crude

product was purified over si li ca co lumn usin g 2 % MeOH in CI-ICI .\ as e luent, yield 0. 73 g (67 %).

General procedure for' the pr'eparation of 9 (Zr dep,'otection) Compou nd 8 was taken in anhydrous trilluoroacetic ac id ( 10 times) and cooled to 0 "c. Ani so le (1.25 eq) was added, foll owed by the addition of tri n uorolll ethanes ulph on ic ac id (I .25 eq). The reac ti on Illi xture was stirred at the sa me temperature for I hr. After the reaction,was over, dry ether was add ed to precipitate the co mpound . The crude product was puri fied by repeated crysta II isati on from MeOI-l­ether. The compound was isolated as trifluoromethanesulphon ic ac id sal t, yield 60 %.

General pmcedure for the preparation of 10 (hydrolysis of 8). Compound 8 was dissolved III

MeOH ( 10 mL), IN NaOH ( 1.0 eq) added to it and the reaction mi xture stirred for I hr at room temperature. After th e reac tion was over, methanol was removed in vacuulll and the aqueous layer extrac ted with CHCb to remove impu riti es. The aqueous layer was then ac idifi ed by IN Hel to p H 6.0 and then extracted with CI ICI,; . The organi c layer was dried over Na2S04 and concentraed to dryness in vacu ulll. The product was used as such for deprotect ion reaction .

General p,'ocedt/l'c fo r the prepa ration of 11. The same procedu re which was used ror Z2 deprotection of 8 (see genera l preparat ion of 9) was uscd to prepare II . Synthesis of C-temlinaJ analogues of 5

Prcpanl tion of 12. Hydro lys is o f 5 to get 12 was achieved using th e sa me procedure as ck scribec1 for th e hydrolys is of 8.

~ .

-

RAMAKR ISHNA el al.: SYNTHESIS OF RGD ANALOGUES OF 2,5-DIK ETOPIPERAZINE 1337

General procedure for the preparation of 13. Condensation of 12 with various ami no-esters was carried out using the procedure described for the preparation of 8. The crude product was purified over si lica column using 5% MeOH in CHC I.1 as eluent, yie ld 60 %.

Preparation of 14. The Z-deprotection of 13 was carried out using the same procedure as described for the Z-deprotection of 5 (preparation of 7), yield 75 %.

Preparation of 15. Compound 14 (0 .7 g, 4.16 mmole) was taken in dry CH 3CN (50 mL), and dry triethylamine (2 .33 mL, 16.64 mmole) added to it. To this Zr S-methylisothiourea (0.62 g, 1.0 eq.) was added in CHjCN (5 mL) over the period of 20 min. The reaction mixture was refluxed overnight. After the reaction was over, the so lvent was removed , res idue diluted with water and extracted with chloroform . The organic layer was dried over Na2S04 and the crude puri fied over si I ica column using 10 % MeOH in CHC I.1 as eluent. The product was subjected to deprotection of carbobenzoyloxy groups on guanidine, using the same procedure as described earlier for the preparation of 9. Compound 15 was isolated as trifluoromethanesulphonic acid sa lt, yield 60%.

Preparation of 16. Compound 15 was hydrolysed to yie ld 16 lIsing the same procedure as described for the preparation of 10 . Pharmacological evaluation

Measurement of platelet aggregation in vitro using human PRP. Platelet aggrega tion was studied by using the method of Born ct 0/.

11. Blood sample

from normal human volu nteer was co ll ected using acid-c itrate-dextrose (ACD) as anticoagulant. This blood was centrifuged at 120 G for 15 ll1in and platelet rich plasll1a (PRP) thus separated was co llected. The remainder blood was further centrifuged at 1000 G for 15 min and platelet poor plasma (PPP) was col lected. Platelet aggregation was measured using a DTC aggrecorder- II. Zero - 100 % transmission adjustments on the aggregometer were

made using PRP and PPP respectively. Plate l ~t

aggregation was induced in PRP by adding ADP at a final concentration of 10 flM. The resultant aggregation was monitored for 1 min.

To study the effect of a test compound on platelet aggregation, PR P was incubated at 37 °C with the test compound at the required concentration for 3 min and then ADP was added. The platelet aggregat ion was recorded for the next 3 min. Maximull1 contro l aggregat ion was taken as 100 % and the per cent aggregat ion in the presence of test compound was calculated. The effect of the compound was expressed as per cent inhibition of aggregation.

Acknowledgements Authors wish to thank Dr P.K. Inamdar and his

group for the ana lytica l data and Dr S. Chatterjee for NMR data and Dr A.K. Gangopadhyay for valuable discussions.

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