Indian Journal of Chemistry Vol. 40B, February 2001 , pp. 1 13- 1 19

Synthsis of hemin and porphyrin derivatives and their evaluation for anticancer activity

Sham M Sondhi*, Nidhi Singhal & Rajeshwar P Verma Department of Chemistry, University of Roorkee, Roorkee 247 667 (U.P.) India

and Sudershan K Arora & Sunanda G Dastidar

Ranbaxy Research Laboratories, Delhi 1 10 020, India

Received 10 November 1999; accepted (revised) 10 August 2000

N.Ethylaminoadenosine, histamine, 2-amino-2-thiazoline, 4-aminoantipyrine, sulphathiazole and a number of 3,4-diaryl-2-iminothiazolines are coupled with hemin to give bis coupled products 3a, 3b, 3c, 3d, 3e and 3f-m, respectively. Mono coupling of 2-amino-2-thiazoline with hemin gives isomeric mixture of mono coupled product 4. Deuteroporphyrin IX dicarboxylic acid is coupled with 2-amino-2-thiazoline to give bis coupled product 6 which on treatment with MnCI2. 4H20 gives compound 7. Compounds 3a·m and 4 have been screened for anticancer activity against a small panel of six cancer cell l ines consisting of prostate tumour (DU 145, PC3), colon carcinoma (HT29 or SW620), melanoma (SK-MEL-5, LOX), breast cancer (MCF 7 and adriamycin resistant MCF 7), CNS (U25 1 ) and ovarian cancer (IGROV I ). Best GI50 (con­centration which inhibits the cell growth by 50%), values are shown by 3f, 6.3 11M (prostate tumour, cell line DU 145); 3r, 6. 1 11M (colon tumor, cell line HT29); 4, 2.09 11M (colon tumor, cel l line SW620); 3f, 2.2 J.lM (melanoma tumor, cell line LOX); 3i, 4.4 J.lM (breast tumor, cell line MCF7/ADR); 3

j, 2.68 11M (ovarian tumor, cell l ine IGROV 1) and 3g, 1 .25 11M

(CNS tumor, cell line U 25 1 ) respectively.

Hemin and metalloporphyrins are very versatile com­pounds known in nature. They have the ability to carry out numerops functions 1 in a free state or in as­sociation with specific proteins, for example hemin inhibited the lipid peroxidation2 and could produce partial recovery to near normal levels3 of hematopoi­eses caused by AZT. Hemes form the prosthetic groups of a number of proteins and these hematopro­teins exhibit impressive range of biological func­tions4. Iron porphyrins5 are a component of hemoglo­bin, myoglobin peroxidases, catalases and cyto­chromes. Metal porphyrin oligonucleotide conjugates6 have been used as chemiluminescent DNA probes. Porphyrin derivatives 7 and Pt(ll) porphyrin com­plexes8 derived from hemin have been used in photo­dynamic therapy which is based on the ability of some porphyrins and porphyrin like chromophores9 to be accumulated selectively in tumor tissues. Tumor ne­crosis can be obtained by irradiation of neoplastic area with light of appropriate wave length. Uptake of radiolabelled porphyrins by tumor is not blocked 10 by hemin and its preinjection induced higher tumor up­take in rats, up to a factor of 5 with 24 hr post­injection. Pretreatment of the animals with hemin caused better therapeutic ratios for porphyrin.

Haemin-acridines showing antileukemic proper­ties " , metalloporphyrins showing tumor growth in­hibitory effect ' 2, porphyrin (Fe) intercalator causing DNA scission13, hematoporphyrin glycoside deriva­tive'4 useful in photochemotherapy of malignant tu­mours and solid phase synthesis of protohemin (IX) peptide derivatives '5 have been reported in literature. The importance of porphyrins and metalloporphyrins has increased significantly over the last decade '6. In the clinic most activity has focused on photodynamic therapy of cancer, porphyrias and hematol, diseases and various forms of jaundice. A recent report indi­cated that propionic acid side chains of hemin 17 play an important role in the induction of erythroid differ­entiation of K562 cells. In continuation of our studies on porphyrins and metalloporphyrins l 8, I9,20 we have synthesized a number of hemin and porphyrin deriva­tives and evaluated for anticancer activity which we wish to report in this paper.

N-Ethylamino adenosine 2a was synthesized by following the procedure reported in literature2 1 , Vari­ous coupling agents i.e, 1 ,3-dicyclohexylcarbodiimide (DCC), 1 , l '-carbonyldiimidazole, 1 -(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were used for the bis coupling of N-ethyl-

1 14 INDIAN 1. CHEM .. SEC. B. FEBRUARY 2001

aminoadenosine with hemin. Best results were ob­tained by using EDC. Therefore, in the synthesis of 3a-m (Scheme I) and 4 (Scheme II) EDC was used

as a coupling agent. Thus hemin and EDC in the ratio of 1 :2 mol equivalent were taken in dry DMF and stirred at room temperature for 2 hr and then N-

CH=CHZ

(I) EDCIDMF stirrng at RT for 1 -�h (II) H-R Stirring at RT for 72h

CH3 (2a-ml H3C

R for 2a-m is sarre as 3a-m

3a R =

1 ctNH�NH-N I """'-:N

J N Ho�OI H�H 3b R ; LL�H-

H

3c R = CJlNH-M�H-

3d R = AN� Me 1h .

3e R = �I� �-<Qr �S RNH-S H-

II o

P\� /Ph 3f R - I J -

S �N-

3h R =

3i R =

3j R =

3k R =

31 R =

3m R =

Scheme l

1HZ �OR

I

5

SONDHI et al. : SYNTHESIS OF HEMIN & PORPHYRIN DERIVATIVES

CH=CHZ

(I) EDCIDMF stirring at RT for Zh •

(II)2c Stirring at RT for 72h

CH=CH2

C� C�

(1) l , l -Carbonyldiimidawlel

DMF stirring at RT for 4h (II) 2c Stirring at RT for 60h

I H2

1H2

O=�"NJCJ H

S

7

Scheme II

'fHZ

tORZ

Rl = OH ; R2 =�S"1

+ N�

R.·I =HN'-.,/N

I�S"1 I � ; RZ = OH

MnCI2.4H20/

DMF

6

4

CH3

1 1 5

1 1 6 INDIAN 1. CHEM., SEC. B, FEBRUARY 2001

ethylaminoadenosine 2a (2 mole equivalent) was added and reaction mixture was stirred at room tem­perature for 72 hr. Removal of solvent under reduced pressure and washing the residue with 1 0% aq. so­dium carbonate solution removed excess EDC. The crude product was crystall ized from methanollDMF to give bis coupled product 3a in 60% yield. (Scheme I). IR spectrum of 3a shows absorption bands at 3432, 34 1 3 and 3249 cm" corresponding to -NH- and -OH functional groups, absorption bands at 1 620 and 1 420 cm" correspond to amidic and aromatic groups. Due to bulky nature of hemin derivatives synthesized, low resolution electron spray (LRES) or fast atom bom­bardment technique (FAB) was used for recording mass spectrum and in case of 3a LRES MS gave M+ ion peak at 1 200.4 which was in agreement with the structure assigned to 3a. In case of compound 3b his­tamine dihydrochloride was first converted to free histamine by using sodium carbonate and the coupling was done in a similar way as for 3a. In case of 3b crude product was purified by column chromatogra­phy over neutral alumina and elution with CCI4: MeOH ( l : I ) gave pure product 3b (Scheme I). LRESMS gave M+ ion peak at 802.3 which was in agreement with the structure assigned to 3b. Similarly 2-amino-2-thiazoline 2c, 2-amino antipyrine 2d and sulfathiazole 2e were coupled with hemin to give cor­responding bis coupled products i .e. 3c, 3d and 3e respectively (Scheme I; Table I)

Various 3,4-diaryl-2-iminothiazolines 2f-m used for the synthesis of 3f-m were synthesized by the condensation of phenacyl thiocyanate and amine hy­drochlorides as reported in literature22. 3,4-Diaryl-2-iminothiazolines 2f-m were coupled with hemin in similar way as for 3a and the crude products were purified by column chromatography over neutral alu­mina to give pure bis coupled products 3f-m (Scheme I). Solvent of elution, m.p. , yield and LRES or FABMS data of 3f-m are reported in Table I. Cou­pling of hemin 1, EDC and 2-amino-2-thiazoline 2c in equimolar ratio gave mono coupled product 4 (Scheme II) which was purified by column chroma­tography over neutral alumina and elution was done with CCI4: MeOH ( 1 : 1 ) to give isomeric mixture of mono coupled product 4. Yield, m.p. and spectral data of 4 are reported in Table I.

Deuteroporphyrin IX dicarboxylic acid 5 was syn­thesized from hemin by following the procedure re­ported in literature23 . Compound 5 was coupled with 2-amino-2-thiazoline 2c by following the procedure reported earlier by us20 to give coupled deuteropor-

phyrin 6 (Scheme II). Coupled deuteroporphyrin i .e. 6 was converted to metalloporphyrin (7; Scheme II) by heating with MnCI2.4H20 in DMF. The structure of 7 was confirmed by spectroscopic means and spectral data of 7 are reported in Table I.

Anticancer activity evaluation of 3a-m and 4 was carried out against a small panel of six cancer cell l ines consisting of prostate (DU 1 45, PC3); colon (HT29, SW620); melanoma (LOX, SK-MEL-5); breast (MCF7, MCF7/ADR); ovarian (lGROVI ) and CNS (U25 1 ) tumors. The effect of various com­pounds screened is expressed in terms of 50% growth inhibitory concentration (G1so) . The GIso values in J.lM concentration of 3a-m and 4 are reported in Table II. From Table II i t is clear that best GIso values are shown by 3f, 6.3 J.lM (prostate, DU 1 45) ; 3f, 6. 1 J.lM (colon, HT29);4, 2.09 J.lM (colon, SW620); 3f, 2.2 J.lM (melanoma, LOX); 3i, 4.4 J.lM (breast, MCF7/ADR); 3j, 2.68 J.lM (ovarian, IGROV I ) and 3g, 1 .2 J.lM (CNS, U25 1 ) tumors respectively. From GIso values of 3a-m it is clear that unsubstituted thiazoline de­rivative i .e. 3f gave good results against prostate, co­lon and melanoma tumors and substitution in the phenyl group l inked with nitrogen of thiazoline by electron donating group prove beneficial for activity against ovarian tumor (in case of 3j) and CNS tumor (in case of 3g) whereas substitution by electron with­drawing group made these compounds less active (as in case of 3h, 3k and 31) than 3f.

Experimental Section

Melting points were determined on JSGW appara­tus and are uncorrected. Only principal sharply de­fined IR peaks are reported. 'H NMR spectra were recorded in ca 5- 1 5% (w/v) solution in an appropriate deuterated solvent with tetramethyl silane as internal standard using a Varian XL-300 spectrometer. Line positions are recorded in ppm from the reference. The MS spectrometer peak measurement were made by comparison with perfluorotributylamine using AEI MS-9 double focusing high resolution mass spec­trometer at a resolving power of 1 5000. TLC was per­formed on silica gel G for TLC (Merk) and spots were visualized by iodine vapour or by irradiation with UY light (254 nm). Column chromatography was per­formed by using neutral alumina or Qualigens silica gel for column chromatography (60- 1 20 mesh).

Coupling of N-ethylaminoadenosine with hemin 3a. Hemin (652 mg; 1 mmole) was taken in dry DMF (20 mL) and to it was added EDC (384 mg; 2 m moles). The reaction contents were stirred at room

Compd

3a

3b

3c

3d

3e

3f

3g

3h

31

3j

3k

31

3m

4

7

SONDHI et al. : SYNTHESIS OF HEMIN & PORPHYRIN DERIVATIVES

Table I-Physical constants and spectral data of hemin 3a-m, 4 and porphyrin derivatives 7

Solvent of elution/ crystallization*

MeOHlDMF·

CCVMeOH ( 1 : I )

CCI4/MeOH ( I : I )

CHCIYMeOH (9: I )

CCVMeOH ( 1 : 4)

CHCliMeOH (9: I )

CHCIiMeOH (9: 1 )

CCliMeOH ( 1 : I )

CCliMeOH ( 1 : I )

MeOH

CHCliMeOH ( 19: 1 )

CHCIYMeOH (9: I )

CHCliMeOH ( 19: I )

CCI4/MeOH ( I : I )

*THF

>260

>260

>270

>260

>260

>260

230(d)

1 80(d)

1 90(d)

>260

>260

250(d)

>250

>260

>260

Yield (%)

60

20

30

20

1 5

35

1 3

25

20

16

1 5

10

20

10

65

LRES or FAB MS(rn/z, relt. Int. %)

LRES:Found: 1200.4 (M+, 1 2) Calcd: for CSSH64NI60JQFe: 1 200.4

LRES:Found: 802.3 (M+, 1 00) Calcd. for C44H46N IOOzFe : 802.3, 735

� I- • �r-.H2 (M+- 9 ,6), 69 1 .2(M+ - �J ,7)

H

LRES: Found: 784.3 (M+, 100) Calcd.for C4oH44NsSzOzFe:784.2

LRES: Found: 986.3 (M+, 100) Calcd for Cs6Hs4N IO04Fe: 986.4.

LRES: Found: 1 090.2 (M+, 30) Calcd. For CSZH46N IO06S4Fe: 1090.2, 836 (M+ .

FABMS: 1084 (M+,40) 279( , 100.00)

+ FABMS: 1 1 1 2 (M+, 60) 293( Ph--rN--<Q>--CH3 , I 00.00) ��A + s N-C=O LRES: Found: 1 174.4 (M+,52) Calcd.for C64HsoN IO06SZFe: 1 1 74.3, 1097

(M+-C6Hs, 1 3) 1052 (M+-C6 H4 NO],+' 30)

FABMS: 1202(M+, 1 00) 1 066(M+-C7H6NO], +. 47).

FABMS: 1 140 (M+, 37)

LRES: Found: 1 143.9 (M+, 100) Calcd. for C66Hs6Ns04SzFe: 1 144.3. 1 036.6 (M+·C7H70 , 35)

Ph--rr- N ---.!()\-CI 867 (M+- �� ... I -� ,5)

sAN FABMS : 1 2 12(M+, 40)

FABMS:700.0 (M+, 0.67)

LRES: Found:73 1 .2 (M+, 14) Calcd. for C36H36NsSzOzMn: 73 1 .2

Neutral alumina was used for column chromatography.

1 1 7

1 1 8 INDIAN J. CHEM .• SEC. B. FEBRUARY 2001

temperature for 2 hr and then to i t was added N­ethylaminoadenosine2 1 (620 mg; 2 mmoles). The re­action contents were stirred for 72 hr and then solvent was removed ill vacuo. To the solid residue left be­hind was added 10% sodium carbonate solution (20 mL) and reaction mixture was stirred for half an hour and then filtered, washed with water and air dried to give crude coupled product which was purified by crystallization from MeOH/DMF to give pure bis coupled product 3a. Yield, m.p., and spectral data is reported in Table I.

Condensation of histamine with hemin 3b. So­dium carbonate ( 106 mg; 1 m mole) was dissolved i n distilled water (5 mL) and to i t was added histamine dihydrochloride ( 1 84 mg; 1 m mole). The reaction contents were stirred at room temperature for 3 hr and then solvent was removed under reduced pressure and the reaction contents were completely dried to give free histamine base. Hemin (325 mg; 0.5 m mole), EDC ( 192 mg; 1 m mole) and dry DMF (20 mL) were stilTed at room temperature for 4 hr and then this so­lution was added to the flask containing histamine free base. The reaction contents were stirred at room temperature for 68 hr and then solvent was removed ill vacuo. To the residue left behind was added dis­tilled water (25 mL) and scratched. The crude solid was filtered, washed with water and air dried. The crude product so obtained was dissolved in DMF ( 10 mL) and adsorbed over neutral alumina. Elution with CCI4: MeOH ( 1 : 1) gave required coupled product 3b. Yield, m.p., solvent of elution and spectral data is re­ported i n Table I.

Condensation of 2-amino-2-thiazoline with he­min 3c. Hemin (652 mg; 1 m mole), EDC (384 mg; 2 m mole) and dry DMF (20 mL) were stirred at room temperature for 2 hI' and then to i t was added 2-amino-2-thiazol ine (204 mg; 2 m moles). The reaction contents were stirred for 72 hr at room temperature. Solvent was removed under reduced pressure and the residue left behind was washed with water and air dried. The crude product was dissolved in methanol and adsorbed over neutral alumina and subjected to column chromatography over neutral alumina. First elution with CCI4: ethyl acetate (4: I ) and ethyl acetate removed side products . Further elution with CC1�: MeOH ( I : 1 ) gave required bis coupled product 3c. Similarly were prepared 3d-m. Yield, m.p . , elution solvent or solvent of crystallization and spectral data are reported in Table I.

Mono coupling 0(' ::!-ami no-2-thiazoi inc with he­min 4. Hemin (652 mg; 1 m mole), EDC ( 1 92 mg; 1

m mole) and DMF (25 mL) were stirred at room tem­perature for 2 hr and then to it was added 2-amino-2-thiazoline ( 106 mg; 1 m mole). The reaction contents were stirred at room temperature for 60 hr and then sol vent was removed under reduced pressure and the solid residue left behind was washed with water and air dried. The crude product so obtained was dis­solved in methanol and adsorbed over neutral alumina and column chromatography was done using neutral alumina. Elution with CCl4 :ethyl acetate (4: 1 ), and ethyl acetate removed side products and further elu­tion with CCI4: MeOH ( 1 : 1 ) gave pure mono coupled product 4. Yield, m.p. and spectral data of 4 are re­ported in Table I.

Bis coupled 2-amino-2-thiazoline deuteropor­phyrin' (IX) derivative 6. Compound 6 was synthe­sized according to the procedure reported by us ear­lier2o.

Synthesis of metalloporphyrin 7. A solution of bis coupled deuteroporphyrin derivative 6 ( 100 mg; 0. 15 m mole) and MnChAH20 ( 120 mg; 0.6 m mole) in DMF ( 10 mL) was heated at 125°C for 1 hr and then solvent was removed under reduced pressure. The solid residue left behind was washed thoroughly with water and then crystallized from THF to give metalloporphyrin derivative 7 in 65% yield. Spectral data of 7 is reported i n Table I.

Anticancer activity screening. Compounds 3a-m and 4 were tested over a broad concentration range (ten-fold dilutions starting from � 100 mM to 10 nM) against six human cancer cell l ines comprised of differ­ent tumor types maintained i n growing condition in RPM! 1640 medium containing 10% fetal calf serum and incubated at 37°C under 5% CO2 atmosphere. All cell l ines were inoculated onto a series of standard 26 well microtitre plate on day zero, followed by 24 hr incubation in the absence of test compound. The in­oculation density used currently in the screen was as per Monk et a1.24• All the test compounds i .e. 3a-m and 4 were dissolved in DMSO and diluted further in cul­ture medium. An aliquot of each dilution was added to the growing cells in 96 well plates and incubated for 48 hI'. After incubation, the assay was terminated by add­ing 50 mL of trichloroacetic acid (TCA) and incubating at 4°C for 30 min. The precipitated cells were washed and staincd with sulphorhodamine B dye for 30 min and the excess dye was washed off with acetic acid . Adsorbed dye was solubilised in Tris base (alkal ine pH) and quanti tated by measuring thc 00 at 490 n 111 in an ELlSA reader. GIso values were calculated accord­ing to Boyd & Paull25 and are reported in Tabk II .

SONDHI et al. : SYNTHESIS OF HEMIN & PORPHYRIN DERIVATIVES 1 1 9

Table II-Anti cancer activity evaluation of hemin derivatives 3a-m and 4

Compd 0150 concentration in flM Prostate" Colon" Melanoma" Breast" Ovarian" CNS"

LOX6 SK-MEL-5b MCF 76 MCF 71 ADRb

GROV lb U25 16

33 37.3 35.9 22.9

3b 65 36.2 37 36.2

3c 28.4 >209.8 24. 1

3d 1 7 .96 1 5.46 1 4.7

3e 16 .61 32.04 96.28

3f 6.3 6 . 1 2.2

3g 1 3.82 > 1 06.8

3h 30.3 1 9.4 1 8

3i 6.6 1 1 .7 1 8.5 1 0.7

3j

> 1 4 1 .4 22.4 32.53

3k 1 1 .48 > 1 00. 1 50.88

31 >67.4 1 1 .98

3m 9.7 1 0.8 6.9

4 1 1 .65 54.65 2.09

"Tumor type, b Cell lines

Acknowledgement We are thankful to Head, RSIC, CDRI, Lucknow,

Prof. J.W. Lown, Department of Chemistry, Univer­sity of Alberta, Edmonton, Alberta, Canada, and tech­nical staff of Chemistry Department, University of Roorkee, Roorkee for FABMS, LRES and IR spectra. Financial help from CSIR, New Delhi (R.P. Verma) and from UGC, New Delhi (Nidhi S inghal) is grate­fully acknowledged.

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