Synthesis of Some New

FABAD J. Pharm. Sci., 26, 119-123, 2001

RESEARCH ARTICLES /BİLİMSEL ARAŞTIRMALAR

Synthesis of Some New

Aminoalkylguanidine Derivatives and Their In Vitro Antimicrobial Activities

Ünsal ÇALIŞ*', Meriç KÖKSAL *,Meral ÖZALP**

Syntlıesis of Some New Aminoalkylguanidine Derivatives and Their ln Vüro Antimicrobial Acti.vities

Summary : in this study, the antimicrobial activities of sev- en compoıınds of aminoalkylguanidine derivatives were test- ed. Five of these conıpounds (Compound 1-5) were syn- thesized previoıısly. Two of the compounds included in this study to evaluate antinıicrobial ejficiency (Compound 6,7) were synthesized originally by the reaction of S- methylthiourea and appropriate amines. The structures of these two conıpounds were confirnıed by JR, ¹ H-NMR and elementary analysis. The antimicrobial activity of ull of the

conıpounds was investigated by broth microdilution method by using two Gram positive (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212) and two Gram negative (Escherichia coH ATCC 25922, Pseudomonas ae- ruginosa ATCC 27853) bacteria and yeast-like fungi (Can- dida albicans ATCC 90028, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019). Among tlıe compounds tested N-[3-(2-Adamantylamino )1-propyl}guanidine sulfate showed the nıost favorable activity.

Key Words: Aminoalkylguanidine, synthesis.. anti-

Received Revised Accepted

microbial activity, antibacterial activity, anti- fungal activity

23.2.2001 21.5.2001 29.5.2001

INTRODUCTION

It is well known that an exarnination of the structures of rnany cornrnercially available antihypertensive drugs reveals a cornrnon functionality in the gua- nidine group. This group is present in cornpounds representing diverse classes of antihypertensives, in- cluding adrenergic neuron blockers (guanethidine), central

az

Bazı Yeni Aminoalkilguanidin Türevlerinin Sentezi ve in Vitro Antimikrobiyal Aktiviteleri

Özet: Bu çalışmada 5 tanesi (Bileşik 1-5) önceden sentezi

gerçekleştirilmiş olan 7 adet anıinoalkilguanidin türevi bi-

leşiğin antimikrobial etkinliği araştırılmıştır. Çalışnıaya katılan bileşiklerden ikisi ( Bilqik 6, 7) S-metiltiyoüre ve uygun aminlerin reaksiyonu ile orijinal olarak sentezlenmiş

ve bu yeni bileşiklerin de yapıları IR, 1 H-NMR ve elemente!

analiz ile aydınlatılmıştır. Bileşiklerin antimikrobiyal ak- tiviteleri ise ikisi 'Gram pozitif (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212) ve ikisi Gram negatif (Escherichia cali ATCC 25922, Pseudomonas ae- ruginosa ATCC 27853) olmak üzere dört çeşit bakteri ve maya benzeri mantarlar (Candida albicans ATCC 90028, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019) kullanılarak mikrodilüsyon yöntemi ile de-

ğerlendirilmiştir. Deneye alınan bileşikler içerisinde; N-[3- (2-Adamantilamino)l-propil]guanidin sülfat en iyi aktiviteyi

göstermiştir.

Anahtar kelimeler: Anıinoalkilguanidin, sentez, anti-

nıikrobiyal aktivite, antibakteriyai aktivite, antifungai aktivite

facine), o:1 antagonists (prazosin), vasodilators (mi- noxidil) and diuretics (amiloride, triamterene), in ad- dition to antibiotics (dihydrostreptomycin suHate) and antiseptics ( chlorhexidine). This widespread na- ture of guanidine functionality is well docurnented in the literaturel-B. It has also been reported that differ- ent guanidine derivatives (I) have analgesic⁶ and an- tirnicrobial activities6•9-15. Thus, dihydrostreptornycin sulfate (II) and chlorhexidine (III), which have anti-

*

**

°

Ç~, Köksal, Özalp

septic activities, contain guanidine rnoiety in their structures as shown (Fig. 1).

;---,

' :-ııı '

: il :

coı-:>111-t.:-NH,

/-""N~...:~---l f 1• \'1.-Nıı 1 Rt'ır·"'~:-ıT ·

\ ,_, I

(1) (il)

(ili)

Figure 1. Structures of some antimicrobial agents con- taining guanidine moiety.

in view of this fact,we have synthesized several corn- pounds having the guanidine functional group to in- vestigate their antirnicrobial activities. in this present study, the antirnicrobial activity of seven cornpounds (Table 1) were tested, five of these cornpounds (Corn- pound 1-5) were synthesized previously to evaluate their antihypertensive activities^8. These cornpounds (Cornpound 1-5) having been synthesized again to check their antirnicrobial activity, according to the rnethod used previousiy8, while two of the corn- pounds (Cornpound 6,7) were synthesized specif- ically for this study. The seven arninoalkylguanidine

Table l. Yields and rnelting points of the eornpounds.

Compound number R Yield (%) M.P.(°C)

l:Jh-fCll~)<-0-IClb,- 93 105-7

2

J:i'"''"'·-

3 lQt..:H-ıCH;:),-

94 284-S(dec.)

4 ~cn,-Nıı-(C!l:.ı.,- 93 273-4(dec.)

c63'"-

5 ^{76 276-7}

'

6

o

C1'-(Cll:J,- 91 232-3

7 Qcıı:-Cll:-

~·ıı. 89 274-5(dec.)

derivatives used in this study, had not been evalu- ated frorn the rnierobiologieal aspec! previously in ^lit- erature.

EXPERIMENTAL SECI10N

Chernistry

Ali chernicals used in this study were supplied by E.

Merek (Darrnstadt, Gerrnany) and Aldrieh (Stein- heirn, Gerrnany). Melting points were deterrnined on a Thornas Hoover Capillary Melting Point Apparatus (Philadelphia, P A, USA) and were uncorrected. ^ln- frared (IR) spectra were reeorded on a Perkin-Elmer FT-IR spectrophotorneter 1720 X (Überlingen, Ger- rnany) as Nujol mulls, liquid filıns or KBr disc (y, cm-1).

1H-NMR spectra in DMSO-d6 were recorded on a Bruker AC 80 MHz spectrophotorneter (Karlsruhe, Germany) by using tetramethylsilane (TMS) as an in- ternal standard (chernieal shift in li, ppm). Elemental analyses were perforrned by the Perkin-Elmer Model 240C and Leco CHNS-932 (Überlingen, Gerrnany) at The Scientific and Teehnical Researeh Council of Tur- key and were within ^± 0.4 % of the theoretical values.

The purity of the eompounds was assessed by TLC on silica gel HF 254 +366 (E. Merek, Darrnstadt, Ger- rnany).

The eornpounds were synthesized according to the well-known procedure of guanidine functionality by

!he reaetion of amine and S-rnethylthiourea5,6,8. N- Substituted amines lc-4c which were used as starting materials in guanidine synthesis were prepared by re- fluxing of the appropriate amine or alcohol and ac- rylonitrile and then reduetion with LiAH416,17. ⁱⁿ eontrast, compound 5d was synthesized by using

Çalış and et. al's rnethod^8. Figure 2 shows the syn- thesis pathway of the eompounds.

General procedure lor ^tlıe preparation of N-{3- (Substituted)-1-propyl] guanidine sulfate (Corn- pounds 1-7)

To a solution of appropriate amine (lc-4c, Sd, 6a, 7a) (0.05 rnol) in 50 mi of water, S-rnethylisothiourea hemisulfate (0.025 mol) was added at room tem- perature. The rnixture was heated at 50°C for 5 h un- der a eontinuous nitrogen purge. The existing gases

FABAD J. Pharm. Sci., 26, 119-123, 2001

L'lı,-ocıı, .. -oıı (la)

N.~ıcıırcrı-cN

'

ıı-x-nı:<:H;C~

(ll•-lh)

ı l.ıAl/lı 1

l

(k-fr)

IJ,.•-<Cf!!ıt-Nll, (;\'.•)

1 l IUJO<.lhl'h

t

1 J,N-iCH, ıı-Nı ~-l:-( ı-n ı,- Plı

(~h)

ı:e

o

u-((.'lf,_ı.d•llı o ^(6•)

l

d

, Nllı

lı-X-CllıCl!ı(_'ll_rNll--"t N!l Hı"l.ı

Com~ound 1-!' X_..0-or-NH-

N!J~

Figure 2. Synthesis of the cornpoundsl-7.

Oımpo1und6

were passed through two flasks, each containing a saturated ethanolic solution of KOH to trap the CH₃SH by product. The rnixture was then con- centrated anda suitable arnount of acetone was add- ed. The precipitate was filtered and recrysta!lized frorn water to give the !itle cornpound .

Microbiology

Minimal inhibitory concentrations (M!Cs) were de, terrnined by broth rnicrodilution rnethod following the procedures recornrnended by the National Corn- mittee for Clinical Laboratory StandardslB,19. Two Gram positive (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212) and two Gram negotive (Escherichia cali ATCC 25922, Pseudo- monas aeruginosa ATCC 27853) bacteria were lJ.Sed as quality control strains. For testing anttfungal activ- ities of the cornpounds, these reference strains were tested: Candida albicans ATCC 90028, Candida kru- sei ATCC 6258, Candida parapsilosis ATCC 22019.

Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) was used when testing bacterial strains.

For Candida species, RPMI-1640 rnediurn with L-

Glutamin, buffered with MOPS (ICN, FLOW; Au- rora, OH, USA) was used. The inocuium densities were 5 x ıos cfu/rnl for bacteria and fungi, re- spectively. The compounds under the test were dis- solved in 100 % dimethylsulfoxide and the final two fold concentrations were prepared frorn 512 µg/ rnl to 0.5 µg/rnl. Arnikacin and Fluconazole were used as reference powders for bacteria and fungi, re- spectively. Two fold dilutions were prepared from 64 µg/rnl to 0.0625 µg/rnl for each of these antibiotics.

MICs were deterrnined after incubation for 24 h at 35°C. Minimal inhibitory concentrations were de- fineci as the lowest concentrations of the anti- rnicrobial agents that inhibited visible growth of the rnicroorganisms.

RESULTS AND DISCUSSION

Oıernistry

The arninoalkylguanidine derivatives (Table 1) were obtained as outlined in Fig2. The general procedure in fig. 2 is !hat of Maxwell and Ueda at al5,6. It was used successfully to synthesize two novel com- pounds (Cornpounds 6,7) in high yields (>89 %) as shown in Table 1. IH-NMR spectrurn assignrnents for each cornpound were made by cornparison with their previously recorded IH-NMR spectruınsB. in the structure of the guanidine, each nitrogen atom has protones and in this structure, rnaximum delocal- ization of the positive charge is the situation, thus, the absence of NH coupling (in the absence of NH signal) is a consequence of the rate of NH exchange in lH-NMR spectrurns of guanidine derivatives20.

Therefore, NH signal regarding guanidine structure couid not be observed in ¹H-NMR spectrums of the synthesized cornpounds. IR and IH-NMR spectra and elernentary analysis data of the synthesized two novel cornpounds (Cornpounds 6,7) are as following.

N-[3-(2-0xopyrrolidinyl)-1-propyl]guanidine sulfate

" (Cornpofilıd 6)

This was synthesized by the general procedure using 3-(2-0xopyrrolidfnyl)propylarnine and S-rnethylisot- hiourea, and crystallized from water. Yield: 91 %.

Çalış, Köksal, Özalp

M.p.: 232-3 °C. IR (KBr, cm-1) 1675 guarıidinium,

3333 +NH; 1H NMR (DMSO-d_6, ö, ppm) 1.80-2.60 (t, 6H, CH_2), 3.20-3.60 (t, 6H, CH2-pyrrolidine); Ana!.

Cal. for C₈H₁₆N₄0.l/2H₂S0₄ (M.W.:233.26): C, 41.20;

H, 6.87; N, 24.03; Found: C, 41.12; H, 6.91; N, 24.18.

N-[2-(l-Methyl-2-pyrrolidinyl)-1-etlıyl]guanidine sul- fa te (Compound 7)

This was synthesized by the general procedure using 2-(1-methyl-2-pyrrolidinyl)ethylamine and S- methylisothiourea, and crystallized from water.

Yield: 89 %. M.p.: 274-5°C (dec.). IR (KBr, cm_1) 1666

guarıidirıium, 3349 +NH; lH ,NMR (DMSO-d_6, ö, ppm) 1.80-2.60 (t, 4H, CH_2), 3.00 (s, 3H, CH_3), 3.20- 3.60 (t, 6H, CHz-pyrrolidine); Ana!. Cal. for C₈H18N4.

H₂S0₄ (M.W.:268.30): C, 35.81; H, 7.51; N, 20.88;

Found: C, 35.92; H, 7.36; N, 20.73.

Microbiology

Antibacterial activity results of the compounds against Gram positive and Gram negative bacteria are shown in Table 2. When antibacterial activity re- sults were investigated, none of the tested com- pounds were found to be active. The results of screening for antifungal activity of the compounds are reported in Table 3. As the N-[3-(1- Table 2. Bacteriostatic activity results of the com-

pounds (MIC µg/ml.)

Compound no S.aureus E. faecalis E. cali P. aeruginosa

1 >64 >64 >64 >64

2

3 >64 >64 >64 >64

4 >64 >64 >64 >64

5 >64 >64 >64 >64

6 >64 >64 >64 >64

7 >64 >64 >64 >64

Amikacin 2 64 1 4

Table 3. Antifungal activity results of the com- pounds (M!C µg/ml.)

Compound no C. albicans C. krusei C. parapsilosis

1 >64 >64 >64

2

3 2 16 32

4 >64 >64 >64

5 >64 >64 >64

6 8 64 >64

7 >64 >64 >64

Flucanazole 0.25 64 2

adamantylamino)-1-propyl]-guanidine sulfate (Com- pound 2) was not soluble in the 100% dim- ethylsulfoxide solvent used, it was not possible to measure its antimicrobial activity. MIC values showed !hat N-[3-(2-adamantylamino)-1-propyl]-

guarıidine sulfate (Compound 3) was the most ac!ive compound to fluconazole against three fungi used among the tested compounds. N-[3-(2-

0xopyrrolidinyl)-1-propyl]guarıidine sulfate (Com- pound 6) had activites similar to fluconazole against

Candida krusei. N-[3-(2-adamantylamino)l-

propyl]guarıidine sulfate (Compound 3) was more ef- fective than fluconazole against Candida knısei

among the aminoalkylguanidine derivatives, tested.

CONCLUSION

Among the synthesized aminoalkylguanidine de- rivatives, N-[3-(2-adamantylamino)l-propyl] gua- nidine sulfate (Compound 3) were found to have sig- nificantiy high antifungal activity against Candida krusei. We assume that Compound 3 is the most im- portant compound in the series regarding its anti- fungal activity.

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