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European Journal of Medicinal Chemistry 52 (2012) 230e241

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European Journal of Medicinal Chemistry

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Original article

Amino acid, dipeptide and pseudodipeptide conjugates of ring-substituted8-aminoquinolines: Synthesis and evaluation of anti-infective, b-haematininhibition and cytotoxic activities

Kirandeep Kaur a, Meenakshi Jain a, Shabana I. Khan c,d, Melissa R. Jacob c, Babu L. Tekwani c,e,Savita Singh b, Prati Pal Singh b, Rahul Jain a,*

aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, IndiabDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, IndiacNational Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USAdDepartment of Pharmacognosy, School of Pharmacy, University of Mississippi, MS 38677, USAeDepartment of Pharmacology, School of Pharmacy, University of Mississippi, MS 38677, USA

a r t i c l e i n f o

Article history:Received 14 February 2012Received in revised form9 March 2012Accepted 11 March 2012Available online 21 March 2012

Keywords:8-AminoquinolinesMalariaAntimicrobialSynthesisb-Haematin inhibition

* Corresponding author. Tel.: þ91 172 229 2024; faE-mail address: rahuljain@niper.ac.in (R. Jain).

0223-5234/$ e see front matter � 2012 Elsevier Masdoi:10.1016/j.ejmech.2012.03.019

a b s t r a c t

Three new series of 8-aminoquinolines with modifications in the side-chain by conjugation with aminoacids, dipeptides and pseudodipeptides have been synthesized. The synthesized compounds were testedfor in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodiumfalciparum strains, in vitro cytotoxicity in mammalian kidney cells (Vero), in vitro antileishmanial activityagainst Leishmania donovani, in vitro antimicrobial activity and in vitro inhibition of b-haematinformation. The promising compounds were also evaluated for in vivo blood-schizontocidal antimalarialactivity against Plasmodium berghei infected mice. The analogues 55 and 101 produced highest anti-malarial activities, in vitro. Analogues 52 and 59 exhibited promising antileishmanial and broad spec-trum of antifungal activities, respectively.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Malaria is one of the major life-threatening parasitic diseases inthe tropical and subtropical regions of the world. Worldwide, thereare at least 300 million acute cases of malaria and more than 1e2million die each year, mainly due to Plasmodium falciparum infec-tion [1,2]. The malaria problem is further exacerbated by the spreadof drug resistant P. falciparum strains [3e5]. In this context, thedevelopment of safe and effective antimalarial drugs preventingtransmission, in addition to curing patients becomes an importantstrategy towards achieving an effective control of malaria.Currently, primaquine (PQ, 1, Fig. 1), composed of an 8-aminoquinoline (8-AQ) structural framework, is one of the avail-able transmission blocking antimalarial drugs. It displays a markedactivity against gametocytes of all species of Plasmodium affectinghumans, including chloroquine-resistant P. falciparum [6,7].However, the use of 1 is restricted and limited due to its extensive

x: þ91 172 221 4692.

son SAS. All rights reserved.

metabolism (for example, conversion to 2, Fig. 1), and its toxic sideeffects, specifically haemolytic anaemia, particularly in patientshaving glucose-6-phosphate dehydrogenase deficiency [8,9].

The therapeutic index profile of 1 has been improved by struc-tural modification/manipulation of the aminoalkyl side-chainresulting in its decreased metabolic inactivation. As examples ofsuch improvement, several side-chain modified analogues of 1have been synthesized [10e20]. A large number of these derivativeshave displayed improved activity/toxicity ratios compared to 1. Wehave earlier reported that 2-tert-butylprimaquine (BPQ, 3, Fig. 1)exhibit potent blood-schizontocidal antimalarial activity [21]. TheBPQ possibly eliminates a putative oxidative metabolic pathwayknown for several quinoline ring-containing antimalarial drugs,including quinine by the placement of a metabolically stable tert-butyl group at the C-2 position of the ring. We also demonstratedthe ability of 3 to inhibit b-haematin formation, in vitro by forminga complex with haem with a stoichiometry of 1:1, resulting inhaem-induced haemolysis, thereby providing evidence of thebiochemical pathway responsible for its antimalarial action [22].The present study was aimed to design, synthesize and evaluatein vitro and in vivo antimalarial activity, cytotoxicity in the

N

O

NHNH2

N

O

NHNH2

N

O

NHOH

O1. Primaquine (PQ) 2. Carboxyprimaquine 3. 2-ter t-Butylprimaquine (BPQ)

Fig. 1. Structure of 8-aminoquinolines.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 231

mammalian cells, inhibition of b-haematin formation, and in vitroantileishmanial and antimicrobial activity of amino acid, dipeptidesand pseudodipeptides conjugates (Fig. 2) of PQ (1) and BPQ (3).

2. Results and discussion

Earlier, we have reported the synthesis of 8-AQs conjugated toamino acids containing a free terminal amino group [13]. Herein,we report synthesis of 8-AQs conjugates (Series 1, Fig. 2) bycoupling the side-chain amino group of the amino acids througha covalent linkage to the terminal amino group of 8-AQs. We havefound earlier that the attachment of cationic amino acids to 8-AQsenhances the antimalarial activity. Therefore, cationic amino acidslike, lysine (Lys) and ornithine (Orn) were selected to synthesize thetarget compounds. The amino acids were adequately protected atthe a-NH2 and a-CO2H group to allow specific reaction with theside-chain NH2 group. These analogues (9e12, Series 1, Scheme 1)allowed us to observe the effect of both free CO2H and NH2 groupson the antimalarial activity.

The earlier studies from our laboratory have shown that theamino acid conjugates of 8-AQs containing cationic amino acidsexhibit higher activities as compared to those containing anionicand lipophilic amino acids [13,15]. The enhanced activities of 8-AQsconjugatedwith cationic amino acids could be explained by the factthat these compounds possibly accumulate in the acidic foodvacuole of the parasite, increasing their ability to complex withhaem, leading to inhibition of b-haematin (BH) formation. Morerecently, a study on the effects of cationic amino acids on the BHinhibition confirms the importance of basicity [23]. Therefore, inprinciple, an increase in the basicity of conjugates should increasetheir accumulation in the parasitic food vacuole to an even greaterextent, resulting in an increase in BH inhibition and antimalarialactivity. To further explore the scope and validity of this hypothesis,we synthesized cationic dipeptide conjugates of 8-AQs (49e60,Series 2, Fig. 2).

The pseudopeptides are a class of compounds inwhich an amidelinkage is modified by chemical changes such as, modification ofthe carbonyl group of amide bond by reduction, thionation or bychanging the linkage between the consecutive amino acids. In

N

O

NHR1

HN

HN

O

NH2

CO2Hn

N

O

NH HN

R1

O

Series 1 Serie

Fig. 2. General structure of synthesiz

pseudopeptides, amino acid residues can also be linked through a/b/g-CO2H or NH2 groups, thereby rendering themmore resistant toenzymatic cleavage. Most of these modifications led to changes inthe hydrophilic or hydrophobic properties, electronic distributions,and geometric/topochemical structure of the resulting compounds.In addition to the synthesis of dipeptide conjugates, we also plan-ned to evaluate the effect of linkage between 8-AQs and aminoacids on the antimalarial activity and synthesized pseudodipeptideconjugates of 8-AQs (101e124, Series 3, Fig. 2). The syntheticstrategy for pseudodipeptide conjugates involves linking of 8-QAsto anionic amino acids (Asp or Glu) through their side-chain b/g-CO2H group. This is followed by a coupling reaction through their a-NH2 group with cationic amino acids (Lys, Arg, or Orn).

2.1. Chemistry

The reaction of 1 or 3 [21] with the suitably protected D/L-aminoacids 4 in the presence of 1,10-carbonyldiimidazole (CDI) in anhy-drous dichloromethane (CH2Cl2) afforded fully protected conju-gates 5e8 in moderate yield (31e42%). The yield of reaction waslow due to the formation of bis-adducts as the by-product in35e40% yield. The complete deprotection of 5e8 was achieved inone or two steps depending upon the nature of the protectinggroups. In cases involving combination of carbobenzyloxy (Z) andmethyl ester groups (compounds 5 and 7), Z group was firstremoved using PdeC/H2 followed by removal of ester group with6 N HCl to provide 9 and 11. In contrast, tert-butoxycarbonyl (t-Boc)and tert-butyl ester protecting groups in compounds 6 and 8 wereremoved in one-step by using 6 N HCl to provide analogues 10 and12 (Scheme 1).

The reaction of 1 or 3 with suitably orthogonally protected D/L-amino acids in the presence of 1,3-diisopropylcarbodiimide(DIC) in CH2Cl2 readily provided carbamates 13e24.The FmoceD-Arg(Pmc)-OH having orthogonal protectinggroups, 9-fluorenylmethyloxycarbonyl (Fmoc) and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) was used for thesynthesis of Arg containing conjugates. Whereas, for all otherconjugates, amino acids with Z as the side-chain amino pro-tecting group and t-Boc as the a-amino protecting group were

NH

R2NH2

O

R2

N

O

NHHN

R1

OnHN

CO2HNH2

R2

O

s 2 Series 3

ed 8-aminoquinoline conjugates.

ii, iii

1. R = H3. R = C(CH3)3 4

5-8

9-12

N

O

NHR

NH2 H2N CO2-tBu/Me

HNi

N

O

NHR

HN

HN

OCO2-tBu/Me

N

O

NHR

HN

HN

O

NH2

CO2H

n = 3 or 4

n

nn

HN

+31-42%

81-95%

Boc/Z Boc/Z

Scheme 1. Reagents and conditions: (i) CDI, DCM, rt, 8 h; (ii) PdeC/H2, MeOH, rt, 4 h; (iii) 6 N HCl, rt, 5 h.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241232

used. All products were obtained in good yields (88e97%),except for D-Arg conjugates, which were obtained in 72e81%yields due to a complex multi-step purification procedure.Depending upon their nature, a-NH2 protecting group in 13e24was removed using acidic or basic conditions. For example, incompounds 13e16, 18e22 and 24, the N-a-Boc group wasremoved by using 4 N HCl in methanol (MeOH), followed byneutralization reaction with the aqueous NH4OH solution togenerate 25e28, 30e34 and 36. On the other hand, the Fmocgroup in 17 and 23 was removed by using 20% piperidine inCH2Cl2 to afford 29 and 35. The coupling reaction of 25e36 withprotected D/L-amino acids in the presence of DIC in CH2Cl2afforded protected dipeptide conjugates 37e48. In the cases of Zgroup containing conjugates 37e40 and 43e46, deprotectionwas achieved by hydrogenolysis with PdeC/H2. It is important tomention that the removal of Z group in Arg containing dipep-tides required a longer reaction time (7 h) and lower yield(74e78%) compared to Lys and Orn conjugates (4 h, 80e85%yield). The acidolysis reaction with 4 N HCl in MeOH cleanlycleaved the t-Boc group of dipeptides 42 and 48. While, dipep-tide conjugates 41 and 47 containing both Z and Pmc groupswere deprotected in two steps. In these cases, Z group was firstremoved (Pd/C-H2) followed by removal of Pmc group by using8 N HCl in MeOH (Scheme 2).

8-Aminoquinolines 1 or 3 upon reaction with the suitableorthogonally protected D/L-amino acids containing a side-chain freecarboxyl group in the presence of the DIC in CH2Cl2 producedcompounds 61e68. The N-a-Fmoc group in 61e68 was removedusing 20% piperidine. The latter compounds 69e76, upon reactionwith the appropriately protected D/L-amino acids in the presence ofDIC afforded fully protected pseudodipeptide conjugates 77e100.The amino and carboxyl protecting groups in the compounds77e100 were removed under different conditions and reagentsdepending upon the nature of the protecting groups. In casesinvolving compounds 77, 79, 80, 82, 85, 88, and 90, both t-Boc andtert-butyl ester groups were easily removed by using 33% HBr inacetic acid in 30 min. However, the application of same strategy forremoving Z and tert-butyl ester groups in a one-step led to anincreased reaction time of 2 h (for analogues 83 and 89) and 6 h (for

analogue 84). We also observed degradation of the product,resulting in lower yield. Therefore, an alternate deprotectionstrategy for compounds 78, 81, 86, 90, 93, 95, 96, 98, and 99 wasadopted. The Z group was cleaved first by hydrogenolysis asdescribed earlier, followed by acidolysis of tert-butyl ester groupusing 4 N HCl. While, for compounds 92, 94, 97 and 100, removal oft-Boc and tert-butyl ester groups was achieved in a one-step byusing 4 N HCl (Scheme 3).

2.2. Biological activity

2.2.1. Antiprotozoal activity, cytotoxicity, and inhibition of b-haematin (BH) formation

Determination of in vitro antimalarial activity was based on theassay of plasmodial LDH activity [24]. The antimalarial activities ofall synthesized analogues are reported as IC50 values againstchloroquine-sensitive (D6) and chloroquine-resistant (W2) strainsofP. falciparum inTables1e3. Invitro cytotoxicityof all the analogueswas determined against mammalian kidney cell line (Vero) up toa highest concentration of 23.8 mg/mL by neutral red assay [25,26].None of the compounds were found to exhibit cytotoxic effectsindicating a selectivity of antimalarial action. The synthesizedconjugates were also assayed for inhibition of BH formationaccording to a procedure reported earlier [27]. The antileishmanialactivities of the conjugates were evaluated in vitro against Leish-mania donovani promastigotes by Alamar Blue assay [28,29].

8-AQs (9e12, Series 1) were either inactive or possessed weakin vitro antimalarial activity. Compound 12 exhibited weak inhibi-tion of the BH formation with an IC50 value of 250 mM. Compounds9e12 were inactive against L. donovani. These results indicate thatthe presence of free CO2H, in addition to the desired free NH2 groupat the terminal position of the 8-AQs is detrimental for anti-protozoal activity (Table 1).

Of all the dipeptide conjugates 49e60 (Series 2), the most activewere 55 (IC50¼ 0.63 and 0.40 mg/mL against D6 and W2 strains,respectively) and 56 (IC50¼ 0.97 and 0.82 mg/mL against D6 andW2strains, respectively) containing L-LyseL-Lys and L-ArgeL-Argsequences, respectively. Analogues containing D-amino acids wereeither inactive or weakly active (IC50¼ 3.3e4.76 mg/mL), except for

N

O

NHNH2

R1 N

O

NH HN

R1

NH

R2

O

N

O

NHHN

R1

NH2

R2

O

N

O

NHHN

R1

NH

R2

O

HN

O

R3

N

O

NHHN

R1

NH

R4

O

NH2

O

R4

i ii

iii

iv

13-24 25-36

37-4849-60

1. R1 = H3. R1 = C(CH3)3

Boc/Z

72-97% 73-90%

82-91%

74-85%

R1 = H/C(CH3)3R2 = Lys(Z)/Arg(Z)2/Arg(Pmc)/Orn(Z)R3 = Lys(Z)/Arg(Z)2/Orn(Z)/Orn(Boc)R4 = Lys/Arg/Orn

Boc/Fmoc

Scheme 2. Reagents and conditions: (i) DIC, Boc/FmocNH-CH(R2)-CO2H, DCM, 0 �Cert, 4 h; (ii) 4 N methanolic HCl, rt, 45 min, 20% NH4OH or 20% piperidine in DCM, rt, 20 min; (iii)DIC, Boc/ZNH-CH(R3)-CO2H, DCM, 0 �Cert, 4 h; (iv) PdeC/H2, MeOH, rt, 4e7 h or 4 N methanolic HCl, rt, 45 min or 8 N methanolic HCl, rt, 8 h.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 233

analogue 53 (R¼H, R1¼ D-Arg), which exhibited IC50 values of 1.3and 1.7 mg/mL against D6 and W2 strains, respectively. Someanalogues inhibited BH formationwith IC50 values ranging between32 and 618 mM. Compounds 51 (R¼H, R1¼ L-Orn), 52 (R¼H,

N

O

NHNH2

R1 N

O

NH HN

R1

O

i

iv

1. R1 = H3. R1 = C(CH3)3

N

O

NH HN

R1

OnHN

CO2HNH2

R3

O

61-6

101-124

n = 1 or 2R1 = H/C(CH3)3R2 = Lys(Boc)/LysR3 = Lys/Arg/Orn

83-91%

76-98

Scheme 3. Reagents and conditions: (i) DIC, FmocNH-CH[(CH2)nCO2H]-CO2 tBu, DCM, 0 �Ce0 �Cert, 4 h; (iv) 33% HBr-CH3CO2H, 0 �Cert, 30 mine6 h or PdeC/H2, MeOH, rt, 4 h or 4 N

R1¼ D-Lys), and 56 [R¼C(CH3)3, R1¼ L-Arg] were most activeagainst L. donovani with IC50 values of 4, 3.5 and 9 mg/mL, respec-tively, while remaining compounds were either inactive ormoderately active (Table 2).

ii

iii

nCO2tBu

N

O

NH HN

R1

OnCO2tBu

NH2

N

O

NHHN

R1

OnHN

CO2tBuNH

R2O

8 69-76

77-100

HN

(Z)/Arg(Z)2/Orn(Boc)

70-79%

83-95%

%

Fmoc

Boc/Z

rt, 4 h; (ii) 20% piperidine in DCM, rt, 20 min; (iii) DIC, Boc/ZNH-CH(R2)-CO2H, DCM,HCl, rt, 5 h.

Table 1In vitro antimalarial (P. falciparum) and antileishmanial (L. donovani) activity, cytotoxicity, and b-haematin (BH) inhibition of 8-aminoquinoliness (9e12) (Series 1).

N

O

NHR

HN

HN

O

NH2

CO2Hn

Compd. no. R n P. falciparum (D6) P. falciparum (W2) Cytotoxicityd (Vero) BH inhibitione L. donovani

IC50a (mg/mL) SIc IC50a (mg/mL) SIc IC50 (mg/mL) IC50 (mM) IC50 (mg/mL) IC90

b (mg/mL)

9 H 4 (L) NAf e NA e NC >1000 NA NA10 H 3 (D) NA e NA e NC >1000 NA NA11 C(CH3)3 4 (L) NA e NA e NC >1000 NA NA12 C(CH3)3 3 (D) 4.2 >5.5 4.76 5 NC 250 NA NAPQ 2.0 >11.9 2.8 >8.5 NC >1000 19.9 NA

Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or W2).a IC50 is the sample concentration that kills 50% cells compared to vehicle control.b IC90 is the sample concentration that kills 90% cells compared to vehicle control.c Chloroquine: IC50 ¼ 0.014 microg/mL, SI¼ 1700 (D6 clone); IC50¼ 0.1 mg/mL, SI¼ 238 (W2 clone). Artemisinin: IC50¼ 0.015 mg/mL, SI¼ 1565 (D6 clone); IC50¼ 0.009 mg/

mL, SI¼ 2644 (W2 clone).d NC, not cytotoxic up to (23.8 mg/mL).e BH inhibition activity: Chloroquine: IC50¼ 80 mM, BPQ: IC50¼ 2.9 mM, PQ: IC50>1000 mM. Antileishmanial activity: Pentamidine: IC50¼ 1 mg/mL, IC90¼ 3.8 mg/mL.

Amphotericin B: IC50¼ 0.19 mg/mL, IC90¼ 0.35 mg/mL.f NA, Not active. “e”, Not tested.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241234

Among the analogues 101e124 (Series 3), 101 (R¼H, R1¼ L-Lys, n¼ 1) emerged as the most potent antimalarial with IC50values of 0.13 and 0.26 mg/mL against D6 and W2 strains,respectively, and it exhibited high selectivity index (SI >91e183).Inhibition of BH formation by analogue 101 (IC50¼ 5.8 mM) was16-fold higher than that of CQ (IC50¼ 80 mM). These results clearly

Table 2In vitro antimalarial (P. falciparum) and antileishmanial (L. donovani) activity, cytotoxicit

N

O

NH HN

R

O

Compd. no R R1 P. falciparum (D6) P. falciparum (W2)

IC50a (mg/mL) SIc IC50

a (mg/mL) S

49 H Lys (L) 2.0 >11.9 2.850 H Arg (L) NA e 4.7651 H Orn (L) NA e NA e

52 H Lys (D) NA e NA e

53 H Arg (D) 1.3 >18.3 1.7 >

54 H Orn (D) NA e 4.7655 C(CH3)3 Lys (L) 0.63 >37.7 0.4 >

56 C(CH3)3 Arg (L) 0.97 >24.5 0.82 >

57 C(CH3)3 Orn (L) 2.5 >9.5 2.0 >

58 C(CH3)3 Lys (D) 4.76 >5.0 2.859 C(CH3)3 Arg (D) NA e NA e

60 C(CH3)3 Orn (D) 4.76 >5.0 3.3PQ 2.0 >11.9 2.8

Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or W2).a IC50 is the sample concentration that kills 50% cells compared to vehicle control.b IC90 is the sample concentration that kills 90% cells compared to vehicle control.c Chloroquine: IC50 ¼ 0.014 microg/mL, SI¼ 1700 (D6 clone); IC50¼ 0.1 mg/mL, SI¼ 238

mL, SI¼ 2644 (W2 clone).d NC, not cytotoxic up to (23.8 mg/mL).e BH inhibition activity: Chloroquine: IC50¼ 80 mM, BPQ: IC50¼ 2.9 mM, PQ: IC50 >

Amphotericin B: IC50¼ 0.19 mg/mL, IC90¼ 0.35 mg/mL.f NA, Not active. “e”, Not tested.

indicate that the potent blood-schizontocidal antimalarialactivity of 101 is probably due to a disturbance in haem catabo-lism pathway in the malarial parasite. Of the remainingcompounds, only 105 (R¼H, R1¼ L-Arg, n¼ 2) and 120[R¼C(CH3)3, R1¼ D-Arg, n¼ 1] possessed moderate antimalarialactivity (IC50¼1.8e2.6 mg/mL). Compound 113 [R¼C(CH3)3,

y, and b-haematin (BH) inhibition of 8-aminoquinoliness (49e60) (Series 2).

NH

R1NH2

O

R1

Cytotoxicityd (Vero) BH inhibitione L. donovani

Ic IC50 (mg/mL) IC50 (mM) IC50 (mg/mL) IC90b (mg/mL)

>8.5 NC 618 NAf NA>5.0 NC 81 NA NA

NC >1000 4 20NC 310 3.5 7

14 NC 32 17 36>5.0 NC 420 40 NA59.5 NC 73 19 3929.0 NC 53 9 3111.9 NC 85 20 NA>8.5 NC >1000 NA NA

NC >1000 20 34>7.21 NC 39 20 38>8.5 NC >1000 19.9 NA

(W2 clone). Artemisinin: IC50¼ 0.015 mg/mL, SI¼ 1565 (D6 clone); IC50¼ 0.009 mg/

1000 mM. Antileishmanial activity: Pentamidine: IC50¼ 1 mg/mL, IC90¼ 3.8 mg/mL.

Table 3In vitro antimalarial (P. falciparum) and antileishmanial (L. donovani) activity, cytotoxicity, and b-haematin (BH) inhibition of 8-aminoquinoliness (101e124) (Series 3).

N

O

RNH H

N

O

NH

CO2Hn

NH2

R1O

Compd. no. R R1 n P. falciparum (D6) P. falciparum (W2) Cytotoxicityd (Vero) BH inhibitione L. donovani

IC50a (mg/mL) SIc IC50

a (mg/mL) SIc IC 50(mg/mL) IC50 (mM) IC50 (mg/mL) IC90b (mg/mL)

101 H Lys 1 (L) 0.13 >183 0.26 >91.5 NC 5.8 NAf NA102 H Arg 1 (L) 3.0 >7.9 2.9 >8.2 NC 120 NA NA103 H Orn 1 (L) e e e e e e e e

104 H Lys 2 (L) NA e NA e NC e NA NA105 H Arg 2 (L) 1.8 >13.2 1.8 >13.2 NC e NA NA106 H Orn 2 (L) NA e NA e NC >1000 NA NA107 H Lys 1 (D) 2.4 >9.9 3.2 >7.4 NC 160 NA NA108 H Arg 1 (D) 3.2 >7.4 4.2 >5.6 NC 210 20.1 33109 H Orn 1 (D) NA e NA e NC >1000 NA NA110 H Lys 2 (D) NA e NA e NC >1000 20.1 36111 H Arg 2 (D) NA e NA e NC >1000 NA NA112 H Orn 2 (D) NA e NA e NC >1000 NA NA113 C(CH3)3 Lys 1 (L) 3.8 >6.2 2.8 >8.5 NC 131 9.4 31114 C(CH3)3 Arg 1 (L) 3.3 >7.2 2.8 >8.5 NC 115 NA NA115 C(CH3)3 Orn 1 (L) 2.7 >8.8 3.0 >7.9 NC 40 20 36116 C(CH3)3 Lys 2 (L) NA e NA e NC >1000 NA NA117 C(CH3)3 Arg 2 (L) 2.3 >10.3 2.0 >11.9 NC 96 NA NA118 C(CH3)3 Orn 2 (L) NA e NA e NC >1000 NA NA119 C(CH3)3 Lys 1 (D) NA e NA e NC >1000 NA NA120 C(CH3)3 Arg 1 (D) 1.8 >13.2 2.6 >9.1 NC 127 NA NA121 C(CH3)3 Orn 1 (D) 2.5 >9.5 3.4 >7 NC >1000 NA NA122 C(CH3)3 Lys 2 (D) NA e NA e NC >1000 NA NA123 C(CH3)3 Arg 2 (D) NA e NA e NC >1000 NA NA124 C(CH3)3 Orn 2 (D) NA e NA e NC >1000 NA NAPQ H 2.0 >11.9 2.8 >8.5 NC >1000 19.9 NA

Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or W2).a IC50 is the sample concentration that kills 50% cells compared to vehicle control.b IC90 is the sample concentration that kills 90% cells compared to vehicle control.c Chloroquine: IC50 ¼ 0.014 microg/mL, SI¼ 1700 (D6 clone); IC50¼ 0.1 mg/mL, SI¼ 238 (W2 clone). Artemisinin: IC50¼ 0.015 mg/mL, SI¼ 1565 (D6 clone); IC50¼ 0.009 mg/

mL, SI¼ 2644 (W2 clone).d NC, not cytotoxic up to (23.8 mg/mL).e BH inhibition activity: Chloroquine: IC50¼ 80 mM, BPQ: IC50¼ 2.9 mM, PQ: IC50 >1000 mM. Antileishmanial activity: Pentamidine: IC50¼ 1 mg/mL, IC90¼ 3.8 mg/mL.

Amphotericin B: IC50¼ 0.19 mg/mL, IC90¼ 0.35 mg/mL.f NA, Not active. “e”, Not tested.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 235

R1¼ L-Lys, n¼ 1] possessed considerable antileishmanial activitywith an IC50 value of 9.4 mg/mL (Table 3).

The results of in vivo blood-schizontocidal antimalarial activityevaluation of selected analogues in Plasmodium berghei mousemalaria model are summarized in Table 4. Analogues 49 and 101

Table 4In vivo (P. berghei) antimalarial activity of selected 8-aminoquinolines.

Compd. no. P. bergheia,b,c

(10 mg/kg/day� 4, oral) (25 mg/kg/day� 4, or

49 2/6 6/651 e e

55 e e

56 e e

57 e e

58 e e

101 4/6 6/6117 e e

120 e e

“e”, Not tested.a The term ‘curative’ indicates complete elimination of malaria parasites from the bodb The term ‘active’ indicates that all of the treated animals show negative parasitaemi

elimination of parasitaemia as indicated by numbers given in parentheses.c The term ‘inactive’ indicates that the treated animals show positive parasitaemia eit

exhibited 100% curative activity (6/6 cures) at a primary test dose of100 mg/kg. While, 51 (OrneOrn conjugate, 4/6 cures), 55 (LyseLysconjugate, 5/6 cures), 56 (ArgeArg conjugate, 5/6 cures) and 120(AspeD-Arg conjugate, 4/6 cures) produced suppressive activityand partial cure; remaining analogues were inactive at 100 mg/kg

al) (50 mg/kg/day� 4, oral) (100 mg/kg/day� 4, oral)

6/6 (6/6) Curativee (4/6) Activee (5/6) Activee (5/6) Activee (0/6) Inactivee (0/6) Inactive6/6 (6/6) Curativee (0/6) Inactivee (4/6) Active

y, and animals survive up to day Dþ 60.a up to Dþ 7. However, by Dþ 60, some mice die, and some survive with complete

her on Dþ 4 or Dþ 7 and usually die by Dþ 14.

Table 5In vitro antibacterial activities of 8-aminoquinolines.

Compd. no. S. aureus MRSA M. intracellulare E. coli

IC50a

(mg/mL)MICb

(mg/mL)MBCc

(mg/mL)IC50

a

(mg/mL)MICb

(mg/mL)MBCc

(mg/mL)IC50

a

(mg/mL)MICb

(mg/mL)MBCc

(mg/mL)IC50

a

(mg/mL)MICb

(mg/mL)MBCc

(mg/mL)

56 ed e e 7 10 20 NAe NA NA NA NA NA59 11.16 20 20 5.96 10 20 NA NA NA NA NA NA60 e e e NA NA NA 10 20 NA NA NA NA113 e e e NA NA NA NA NA NA 15 20 NA114 e e e NA NA NA NA NA NA 15 NA NA

a IC50¼ the concentration (mg/mL) that affords 50% growth inhibition.b MIC, minimum inhibitory concentration (the lowest concentration in mg/mL that allows no detectable growth).c MBC, minimum bactericidal concentration (the lowest concentration in mg/mL that kills the organism).d “e”, not tested.e NA, no activity at the highest test concentration of 20 mg/mL. Ciprofloxacin: IC50¼ 0.12 mg/mL, MIC¼ 0.50 mg/mL, MBC¼ 50 mg/mL (Sa); IC50¼ 0.09 mg/mL, MIC¼ 0.31 mg/

mL, MBC¼ 2.5 mg/mL (MRSA); IC50¼ 0.3 mg/mL, MIC¼ 0.63 mg/mL, MBC¼ 2.5 mg/mL (Mi), IC50¼ 0.003 mg/mL, MIC¼ 0.016 mg/mL, MBC¼ 0.016 mg/mL (Ec).

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241236

with all mice dying by Dþ 14. The analogues 49 and 101 were alsocurative (6/6 cures) at the lower test doses (50 and 25 mg/kg), andexhibited suppressive activity at the lowest tested dose of 10 mg/kgproducing 2/6 and 4/6 cures, respectively.

2.2.2. Antimicrobial activitiesThe conjugates were also tested for their antibacterial properties

against Staphylococcus aureus ATCC 29213, methicillin-resistant S.aureus ATCC 43300 (MRSA), Mycobacterium intracellulare ATCC23068, Escherichia coli ATCC 35218, and Pseudomonas aeruginosaATCC 27853. Susceptibility testing was performed using a modifiedversion of the CLSI (formerly NCCLS) methods [30e34], whileM. intracellulare was tested using a modified method of Franzblauet al. [35].

The antibacterial activities of selected conjugates 56, 59, 60, 113and 114 are reported in Table 5, including the positive controlciprofloxacin. The remaining conjugates were inactive (data notincluded). None of the conjugates was active against P. aeruginosa(data not shown). The analogues 56 and 59 (Series 2) possessedpromising activity against MRSA exhibiting IC50 values of 7 mg/mL

Table 6In vitro antifungal activities of 8-aminoquinolines.

Compd. no. C. albicans C. glabrata

IC50a

(mg/mL)MICb

(mg/mL)MFCc

(mg/mL)IC50

a

(mg/mL)MICb

(mg/mL)MFC(mg/m

49 NAd NA NA ee e e

50 NA NA NA e e e

51 NA NA NA e e e

52 NA NA NA e e e

53 NA NA NA e e e

54 NA NA NA e e e

55 NA NA NA e e e

56 15 20 NA e e e

57 NA NA NA e e e

58 NA NA NA e e e

59 NA NA NA 12.42 20 2060 NA NA NA e e e

107 NA NA NA e e e

109 NA NA NA e e e

113 NA NA NA e e e

114 NA NA NA e e e

115 15 20 20 e e e

116 NA NA NA e e e

117 NA NA NA e e e

118 NA NA NA e e e

121 NA NA NA 13.68 20 20122 NA NA NA NA NA NA

a IC50¼ the concentration (mg/mL) that affords 50% growth inhibition.b MIC, minimum inhibitory concentration (the lowest concentration in mg/mL that alloc MFC, minimum fungicidal concentration (the lowest concentration in mg/mL that kild NA, no activity at the highest test concentration of 20 mg/mL.e “e”, not tested. Amphotericin B: IC50¼ 0.25 mg/mL, MIC¼ 0.63 mg/mL, MFC¼ 1

IC50¼ 0.6 mg/mL, MIC¼ 1.25 mg/mL, MFC¼ 1.25 mg/mL (Ck); IC50¼ 0.75 mg/mL, MIC¼ 1.2

and 5.9 mg/mL, MIC value of 10 mg/mL, and MBC value of 20 mg/mL,respectively. Analogue 59 was also active against S. aureus dis-playing IC50 value of 11.6 mg/mL, and MIC and MBC of 20 mg/mL.Another analogue 60 of the series was active againstM. intracellularewith IC50 of 10 mg/mL, andMIC of 20 mg/mL but wasnot bactericidal. Analogues 113 and 115 (Series 3) were moderatelyactive against E. coli exhibiting an IC50 value of 15 mg/mL, but nonewere bactericidal at 20 mg/mL.

The antifungal activities of the selected conjugates aresummarized in Table 6 along with the positive control amphoter-icin B. None of the conjugates was active against Aspergillus fumi-gatus (data not shown). All of the conjugates 49e60 (Series 2)exhibited good activity against Cryptococcus neoformans with IC50values ranging between 2.5 and 7 mg/mL. The most active analogue56 of the series (IC50¼ 2.5 mg/mL, MIC¼ 5 mg/mL) was also fungi-cidal at 5 mg/mL. The analogues 52, 54, 55, 57 and 60 exhibited IC50values of 3.5 mg/mL, and MIC and MFC of 5 mg/mL. Except for 56(IC50¼10 mg/mL, MIC¼ 20 mg/mL), all other conjugates were inac-tive against C. albicans. Analogue 59 showed moderate activityagainst Candida glabrata and Candida krusei with IC50 values

C. krusei C. neoformans

c

L)IC50

a

(mg/mL)MICb

(mg/mL)MFCc

(mg/mL)IC50

a

(mg/mL)MICb

(mg/mL)MFCc

(mg/mL)

e e e 7 10 20e e e 4.5 10 10e e e 5.5 10 20e e e 3.5 5 5e e e 7.0 10 10e e e 3.5 5 5e e e 3.5 5 5e e e 2.5 5 5e e e 3.5 5 5e e e 6.5 10 107.08 10 10 3.55 10 10e e e 3.5 5 5e e e 4.5 10 10e e e 7 10 10e e e 3.5 5 5e e e 15 20 20e e e 4 20 20e e e 15 20 20e e e 15 20 20e e e 15 20 20NA NA NA 4.86 10 10NA NA NA 15.19 20 NA

ws no detectable growth).ls the organism).

.25 mg/mL (Ca); IC50¼ 0.07 mg/mL, MIC¼ 0.31 mg/mL, MFC¼ 0.625 mg/mL (Cg);5 mg/mL, MFC¼ 1.5 mg/mL (Cn).

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 237

ranging between 7 and 12 mg/mL, and MIC and MFC values in therange of 10e20 mg/mL. Analogues 107, 113, 115, and 121 (Series 3)displayed good activity against C. neoformans with IC50 valuesranging between 3.5 and 4.5 mg/mL, and MIC and MFC values in therange of 10e20 mg/mL.

3. Conclusions

In conclusion, three new series of 8-aminoquinolines modifiedat the terminal amino group have been designed and synthesized.The compounds containing both free amino and carboxyl groupswere less potent than primaquine, possibly due to the presence ofundesirable carboxyl group. The dipeptides having Arg and Lysresidues exhibited promising in vitro and in vivo antimalarialactivity. Interestingly, dipeptide analogues containing D-aminoacids were either inactive or weakly active. The L-Lys containingpseudodipeptide 101 has demonstrated most promising anti-protozoal activities. The synthesized analogues also exhibitedpotent inhibition of BH formation underlying their biochemicalmechanism of antimalarial activity. In the in vivo blood-schizo-ntocidal activity determination against P. berghei infected micemodel, 49 and 101 showed 100% curative activity at a dose of25 mg/kg, and exhibited suppressive activity at the lowest testdose of 10 mg/kg. Some analogues exhibited promising antibac-terial activity against MRSA and M. intracellulare. Majority of thenewly synthesized 8-aminoquinolines were active againstC. neoformans (IC50¼ 2.5e15.1 mg/mL). It can be safely concludedthat newly synthesized 8-aminoquinolines exhibit broad spec-trum of activities against several pathogenic protozoal andmicrobial infections, which will be further explored to provideadditional promising compounds with improved biologicalactivities.

4. Experimental

4.1. Material and methods

The analogues were checked for their purity on pre-coated silicagel G254TLC plates (Merck) and the spots were visualized under UVlight and by exposing them to iodine vapors. Column chromato-graphic purification was carried out on Merck silica gel(100e200 mesh). Melting points were recorded on a capillarymelting point apparatus and are uncorrected. All solvents used forsynthesis were of analytical grade and used without any furtherpurification unless otherwise stated. 1H and 13C NMR spectra wererecorded on a 300 MHz Bruker FT-NMR (Avance DPX 300) spec-trometer using tetramethylsilane as internal standard and thechemical shifts are reported in d units. Mass spectra were recordedon a Finnigan Mat LCQ spectrometer (APCI/ESI). Elemental analyseswere recorded on Elementar Vario EL spectrometer. The elementalanalyses of all final compounds were within �0.4% of the expectedvalues, unless otherwise stated. All reagents were purchased fromAldrich Chemicals Ltd.

4.2. General method for the synthesis of N5/6-({4-[(6-methoxy-2-substituted-quinolin-8-yl)amino]pentyl}carbamoyl)-D/L-lysine/ornithine$2HCl (9e12)

4.2.1. Removal of Z and methyl ester groupsA slow stream of H2 gas was bubbled to a mixture of quinoline

conjugates (5 or 7, 0.1 mmol), 10% PdeC (0.04 g), and glacial aceticacid (1 mL) in CH3OH (20 mL) for 4 h at ambient temperature. Thecatalyst was filtered and solvent removed. To the residuewas added6 N HCl (5 mL) and reactionwas stirred at ambient temperature for5 h. The solvent was removed to afford the product.

4.2.2. Removal of t-Boc and tert-butyl ester groupsA solution of 6 or 8 (0.11 mmol) in 6 N HCl (5 mL) was stirred at

ambient temperature for 5 h. The solvent was evaporated underreduced pressure to afford product as hygroscopic salt.

4.2.3. N6-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-D-lysine$2HCl (9)

Yield: 85%; hygroscopic solid; IR (KBr): 3437, 1728, 1648 cm�1;1H NMR (CD3OD): d 8.96 (m, 2H), 8.01 (m, 1H), 7.05 (m, 2H), 4.03 (s,3H), 3.69 (m, 2H), 3.38 (m, 4H), 1.83 (m, 10H), 1.31 (d, 3H,J¼ 6.3 Hz); 13C NMR (CD3OD): d 172.3, 171.6, 162.9, 148.1, 146.2,141.4, 140.4, 137.5, 134.0, 120.8, 107.1, 97.3, 57.1, 54.2, 42.2, 41.4, 34.2,29.1, 27.7, 26.8, 26.0, 20.1; APCIMS:m/z 432 (Mþ 1); Anal. Calcd forC22H35Cl2N5O4 (503.2): C, 52.38; H, 6.99; N, 13.88. Found: C, 52.46;H, 7.08; N, 13.96.

4.2.4. N5-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-L-ornithine$2HCl (10)

Yield: 95%; hygroscopic solid; IR (KBr): 3410, 1723, 1637 cm�1;1H NMR (CD3OD): d 8.85 (m, 2H), 7.91 (m, 1H), 6.87 (m, 2H), 4.03 (s,3H), 3.85 (t, 1H, J¼ 5.5 Hz), 3.69 (m, 1H), 3.49 (m, 4H), 1.97 (m, 8H),1.36 (d, 3H, J¼ 6.5 Hz); 13C NMR (CD3OD): d 172.0,171.2, 162.9,161.1,148.1, 146.2, 141.4, 140.4, 137.5, 134.0, 127.8, 120.8, 107.1, 57.1, 54.2,42.2, 41.4, 34.2, 29.1, 27.7, 26.8, 26.0, 20.1; APCIMS:m/z 418 (Mþ 1);Anal. Calcd for C21H33Cl2N5O4 (489.1): C, 51.43; H, 6.78; N, 14.28.Found: C, 51.52; H, 6.85; N, 14.39.

4.2.5. N6-({4-[(2-tert-Butyl-6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-D-lysine$$2HCl (11)

Yield: 81%; hygroscopic solid; IR (KBr): 3398, 1663 cm�1; 1HNMR (CD3OD): d 8.21 (d, 1H, J¼ 8.3 Hz), 7.69 (d, 1H, J¼ 8.3 Hz), 7.43(s, 1H), 7.07 (s, 1H), 3.96 (m, 4H), 3.31 (m, 5H), 1.84 (m, 10H), 1.48 (s,9H), 1.38 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 172.0, 169.6, 161.2,160.6, 157.8, 138.2, 135.7, 119.3, 97.3, 92.1, 67.3, 60.4, 57.1, 41.7, 38.7,32.6, 28.1, 27.5, 26.9, 20.8; APCIMS:m/z 488 (Mþ 1); Anal. Calcd forC26H43Cl2N5O4 (559.2): C, 55.71; H, 7.73; N, 12.49. Found: C, 55.82;H, 7.68; N, 12.57.

4.2.6. N5-({4-[(2-tert-Butyl-6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-L-ornithine$2HCl (12)

Yield: 91%; hygroscopic solid; IR (KBr): 3283, 3030, 1640 cm�1;1H NMR (CD3OD): d 8.35 (d, 1H, J¼ 8.7 Hz), 7.82 (d, 1H, J¼ 8.7 Hz),7.58 (s, 1H), 7.47 (s, 1H), 4.09 (s, 3H), 4.03 (t, 1H, J¼ 6.1 Hz), 3.31 (m,5H), 1.98 (m, 8H), 1.50 (s, 9H), 1.40 (d, 3H, J¼ 6.3 Hz); 13C NMR(CD3OD): d 172.0, 169.6, 161.2, 161.2, 158.4, 138.2, 130.7, 122.3, 98.1,92.3, 67.3, 60.4, 57.1, 41.7, 39.7, 32.2, 30.8, APCIMS: m/z 474 (Mþ 1);Anal. Calcd for C25H41Cl2N5O4 (545.2): C, 54.94; H, 7.56; N, 12.81.Found: C, 55.06; H, 7.62; N, 12.91.

4.3. General method for the synthesis of D/L-lysyl/arginyl/ornithyl-N-{4-[(6-methoxy-2-substituted-quinolin-8-yl)amino]pentyl}-D/L-lysinamide/argininamide/ornithinamide$4HCl (49e60)

To a mixture of Z group containing conjugates 37e40 and43e46(0.11 mmol), glacial acetic acid (1 mL) and 10% PdeC (0.04 g) inCH3OH (5 mL) was bubbled a slow stream of H2 gas for 4 h. Thecatalyst was filtered and filtrate was evaporated under reducedpressure. The resulting residue upon treatment with a solution ofethereal HCl (2 N) provided the hydrochloride salts of 49e52 and55e58.

Alternatively, in the cases of conjugates containing Z and t-Bocgroups (42 and 48) and Z and Pmc groups (41 and 47), Z group wasremoved first as described above. The resulting residue was stirredin 6 N HCl in methanol (10 mL) for 45 min (for t-Boc group) or 8 N

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241238

HCl in methanol (5 mL) for 8 h (for Pmc group). The solvent wasremoved to afford 53, 54, 59 and 60 as hygroscopic salts.

4.3.1. D-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-lysinamide$4HCl (49)

Yield: 84%; hygroscopic solid; IR (KBr): 3368, 1690 cm�1; 1HNMR (CD3OD): d 8.86 (m, 2H), 7.92 (m, 1H), 7.04 (s, 1H), 6.91 (s, 1H),4.10 (t, 1H, J¼ 5.7 Hz), 3.86 (s, 3H), 3.81 (m, 4H), 2.99 (m, 4H),1.98e1.74 (m, 16H), 1.36 (d, 3H, J¼ 6.1 Hz); 13C NMR (CD3OD):d 175.4, 175.2, 162.4, 158.3, 147.6, 146.8, 137.9, 137.8, 133.0, 124.4,99.8, 94.6, 57.2, 56.2, 41.8, 41.8, 36.5, 35.2, 29.6, 29.5, 28.6, 25.3, 24.5,22.3; APCIMS: m/z 516 (Mþ 1); Anal. Calcd for C27H49Cl4N7O3(659.2): C, 49.02; H, 7.47; N, 14.82. Found: C, 49.16; H, 7.38; N, 14.97.

4.3.2. D-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-argininamide$4HCl (50)

Yield: 76%; hygroscopic solid; IR (KBr): 3401, 1701 cm�1; 1HNMR (free base, CDCl3): d 9.34 (bs, 2H), 9.20 (bs, 2H), 8.49 (d, 1H,J¼ 4.1 Hz), 8.04 (d, 1H, J¼ 8.2 Hz), 7.38 (dd, 1H, J¼ 4.1 and 8.2 Hz),6.46 (d,1H, J¼ 2.3 Hz), 6.30 (d,1H, J¼ 2.3 Hz), 6.27 (bs, 2H), 6.15 (bs,2H), 5.97 (bs, 2H), 5.34 (bs, 2H), 4.95 (bs, 1H), 4.30 (m, 1H), 3.87 (s,3H), 3.82e3.73 (m, 4H), 3.31 (m, 4H), 1.68 (m, 12H), 1.29 (d, 3H,J¼ 5.5 Hz); 13C NMR (free base, CDCl3): d 170.1, 168.9, 162.0, 145.1,140.2, 139.7, 133.1, 126.9, 122.5, 105.8, 96.4, 62.1, 56.0, 44.6, 43.9,39.9, 33.6, 30.9, 26.3, 22.9, 20.3; APCIMS: m/z 572 (Mþ 1); Anal.Calcd for C27H49Cl4N11O3 (715.2): C, 45.19; H, 6.88; N, 21.47. Found:C, 45.27; H, 7.01; N, 21.38.

4.3.3. D-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-ornithinamide$4HCl (51)

Yield: 85%; hygroscopic solid; IR (KBr): 3467, 1640 cm�1; 1HNMR (CD3OD): d 8.49 (s, 1H), 8.01 (s, 1H), 7.38 (s, 1H), 6.46 (s, 1H),6.31 (s, 1H), 4.34 (m, 1H), 3.87 (s, 3H), 3.61 (m, 1H), 3.31e3.24 (m,3H), 2.91 (m, 4H), 1.93e1.72 (m, 12H), 1.29 (d, 3H, J¼ 5.6 Hz); 13CNMR (CD3OD): d 174.8, 159.8, 144.7, 135.7, 122.3, 97.7, 92.4, 55.0,53.5, 39.5, 34.4, 30.9, 29.6, 26.4, 24.4, 23.9, 22.4, 20.1; APCIMS: m/z488 (Mþ 1); Anal. Calcd for C25H45Cl4N7O3 (631.2): C, 47.40; H, 7.16;N, 15.48. Found: C, 47.32; H, 7.28; N, 15.61.

4.3.4. L-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-lysinamide$4HCl(52)

Yield: 82%; hygroscopic solid; IR (KBr): 3497, 1652 cm�1; 1HNMR (CD3OD): d 8.37 (s, 1H), 7.92 (s, 1H), 7.25 (m, 1H), 6.34 (s, 1H),6.19 (s, 1H), 4.19 (m,1H), 3.76 (s, 3H), 3.51 (m,1H), 3.19 (m, 3H), 2.79(m, 4H), 1.83 (m, 8H), 1.55 (m, 8H), 1.27 (d, 3H, J¼ 6.6 Hz); 13C NMR(CD3OD): d 176.9, 173.3, 160.3, 158.0, 145.5, 144.8, 135.8, 131.0, 122.4,97.8, 92.5, 64.0, 57.7, 55.1, 39.8, 39.6, 34.4, 31.7, 27.2, 26.5, 23.5, 21.9;APCIMS: m/z 516 (Mþ 1); Anal. Calcd for C27H49Cl4N7O3 (659.2): C,49.02; H, 7.47; N, 14.82. Found: C, 48.94; H, 7.60; N, 14.71.

4.3.5. L-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-argininamide$4HCl (53)

Yield: 74%; hygroscopic solid; IR (KBr): 3392, 1639 cm�1; 1HNMR (CD3OD): d 8.80 (m, 2H), 7.92 (m,1H), 7.04 (s, 1H), 6.88 (s, 1H),4.01 (t, 1H, J¼ 5.8 Hz), 3.99 (s, 3H), 3.32 (m, 4H), 2.13 (m, 4H), 1.80(m, 12H), 1.36 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 174.0, 170.4,159.0, 146.3, 141.2, 140.4, 134.0, 123.6, 117.8, 98.2, 91.3, 57.2, 55.3,51.8, 42.4, 42.2, 30.6, 30.0, 27.5, 27.4, 26.8, 25.5, 20.02; MALDIMS:m/z 573 (Mþ2); Anal. Calcd for C27H49Cl4N11O3 (715.3): C, 45.19; H,6.88; N, 21.47. Found: C, 45.25; H, 6.96; N, 21.32.

4.3.6. L-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-ornithinamide$4HCl (54)

Yield: 85%; hygroscopic solid; IR (KBr): 3415, 1653 cm�1; 1HNMR (CD3OD): d 7.61 (m, 3H), 6.72 (m, 2H), 2.89 (m, 1H), 2.77 (s,

3H), 2.62 (m, 1H), 2.40 (m, 3H), 1.79 (m, 4H), 0.76 (m, 12H), 0.15 (d,3H, J¼ 5.7 Hz); 13C NMR (CD3OD): d 172.8, 169.1, 161.9, 145.0, 140.3,139.4, 133.0, 122.5, 106.1, 91.7, 56.0, 53.8, 52.9, 50.8, 39.8, 39.5, 33.5,29.4, 28.8, 26.2, 24.4, 23.2, 21.1; APCIMS: m/z 488 (Mþ 1); Anal.Calcd for C25H45Cl4N7O3 (631.2): C, 47.40; H, 7.16; N,15.48. Found: C,47.40; H, 7.16; N, 15.48.

4.3.7. D-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-lysinamide$4HCl (55)

Yield: 83%; oil; IR (KBr): 3489, 1644 cm�1; 1H NMR (CD3OD):d 8.36 (d, 1H, J¼ 8.5 Hz), 7.82 (d, 1H, J¼ 8.5 Hz), 7.64 (s, 1H), 7.48 (s,1H), 4.04 (t, 1H, J¼ 6.1 Hz), 3.99 (s, 3H), 3.61 (m, 1H), 3.25 (m, 3H),2.98 (m, 4H), 1.74 (m, 16H), 1.50 (s, 9H), 1.40 (d, 3H, J¼ 5.8 Hz); 13CNMR (CD3OD): d 176.4, 174.2, 169.6, 158.4, 138.1, 136.6, 132.7, 130.3,122.3, 108.4, 97.6, 57.2, 54.3, 40.8, 40.4, 32.3, 30.8, 28.4, 28.2, 25.8,24.4, 23.3, 22.9; APCIMS: m/z 572 (Mþ 1); Anal. Calcd forC31H57Cl4N7O3 (715.3): C, 51.88; H, 8.01; N, 13.66. Found: C, 51.73;H, 8.10; N, 13.74.

4.3.8. D-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-argininamide$4HCl (56)

Yield: 78%; hygroscopic solid; IR (KBr): 3445, 1662 cm�1; 1HNMR (CD3OD): d 8.36 (d, 1H, J¼ 8.6 Hz), 8.18 (d, 1H, J¼ 8.6 Hz), 7.35(m, 2H), 4.09 (t, 1H, J¼ 5.5 Hz), 3.99 (s, 3H), 3.60 (m, 1H), 3.35e3.26(m, 7H), 1.99e1.76 (m, 12H), 1.50 (s, 9H), 1.41 (d, 3H, J¼ 6.1 Hz); 13CNMR (CD3OD): d 173.0, 169.4, 168.6, 158.1, 137.2, 129.2, 12.1, 118.3,107.8, 97.6, 56.2, 54.3, 53.2, 41.4, 41.2, 35.6, 31.4, 29.8, 29.0, 25.3,24.5, 22.3, 21.7; APCIMS: m/z 628 (Mþ 1); Anal. Calcd forC31H57Cl4N11O3 (771.3): C, 48.13; H, 7.43; N, 19.91. Found: C, 48.24;H, 7.37; N, 19.86.

4.3.9. D-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-ornithinamide$4HCl (57)

Yield: 83%; hygroscopic solid; IR (KBr): 3331, 1691 cm�1; 1HNMR (CD3OD): d 8.37 (d, 1H, J¼ 8.0 Hz), 7.82 (d, 1H, J¼ 8.0 Hz), 7.64(s, 1H), 7.47 (s, 1H), 4.40 (t, 1H, J¼ 5.9 Hz), 4.00 (s, 3H), 3.69 (m, 1H),3.36 (m, 3H), 3.03 (m, 4H), 1.88 (m, 12H), 1.51 (s, 9H), 1.42 (d, 3H,J¼ 5.1 Hz); 13C NMR (CD3OD): d 174.7, 173.8, 169.5, 158.4, 138.1,136.6, 133.6, 129.0, 121.8, 118.3, 108.0, 57.2, 54.9, 53.9, 40.6, 40.5,32.5, 30.8, 27.0, 25.4, 24.3; APCIMS:m/z 544 (Mþ 1); Anal. Calcd forC29H53Cl4N7O3 (687.3): C, 50.51; H, 7.75; N, 14.22. Found: C, 50.58;H, 7.84; N, 14.31.

4.3.10. L-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-lysinamide$4HCl (58)

Yield: 81%; hygroscopic solid; IR (KBr): 3401, 1692 cm�1; 1HNMR (CD3OD): d 8.07 (d, 1H, J¼ 8.2 Hz), 7.52 (d, 1H, J¼ 8.2 Hz), 7.37(s, 1H), 7.19 (s, 1H), 4.03 (m, 1H), 3.76 (s, 3H), 3.69 (m, 1H), 3.05 (m,3H), 2.69 (m, 4H), 1.66 (m, 16H), 1.21 (s, 9H), 1.11 (d, 3H, J¼ 5.7 Hz);13C NMR (CD3OD): d 171.0, 167.8, 166.9, 155.7, 138.6, 137.9, 127.6,119.6, 108.1, 97.3, 57.2, 54.5, 52.8, 51.6, 43.0, 38.2, 38.0, 37.8, 37.0,30.3, 29.7, 29.6, 28.1, 25.7, 25.5, 21.7; APCIMS: m/z 572 (Mþ 1);Anal. Calcd for C31H57Cl4N7O3 (715.3): C, 51.88; H, 8.01; N, 13.66.Found: C, 52.01; H, 8.10; N, 13.54.

4.3.11. L-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-argininamide$4HCl (59)

Yield: 75%; hygroscopic solid; IR (KBr): 3445, 1683 cm�1; 1HNMR (CD3OD): d 7.81 (d, 1H, J¼ 8.2 Hz), 7.10 (d, 1H, J¼ 8.2 Hz), 6.50(s, 1H), 6.48 (s, 1H), 4.13 (t, 1H, J¼ 6.3 Hz), 3.87 (s, 3H), 3.56 (m, 4H),2.65 (m, 4H), 1.55 (m, 12H), 1.40 (s, 9H), 1.26 (d, 3H, J¼ 6.1 Hz); 13CNMR (CD3OD): d 174.7, 169.0, 163.0, 157.7, 144.1, 134.1, 134.0, 128.3,118.0, 105.4, 96.1, 56.0, 55.7, 43.1, 39.8, 35.8, 31.5, 25.9, 21.6;APCIMS:m/z 628 (Mþ 1); Anal. Calcd for C31H57Cl4N11O3 (771.3): C,48.13; H, 7.43; N, 19.91. Found: C, 48.06; H, 7.51; N, 20.11.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 239

4.3.12. L-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-ornithinamide$4HCl (60)

Yield: 84%; hygroscopic solid; IR (KBr): 3362, 1678 cm�1; 1HNMR (CD3OD): d 7.53 (d, 1H, J¼ 8.2 Hz), 7.16 (d, 1H, J¼ 8.2 Hz),6.62 (s, 1H), 6.47 (s, 1H), 3.21 (t, 1H, J¼ 6.0 Hz), 2.92 (s, 3H), 2.68(m, 2H), 2.31 (t, 2H, J¼ 6.4 Hz), 2.17 (m, 4H), 0.80 (m, 12H), 0.32 (s,9H), 0.22 (d, 3H, J¼ 6.2 Hz); 13C NMR (CD3OD): d 171.6, 167.3,158.4, 143.9, 143.7, 134.2, 128.9, 120.9, 95.9, 90.7, 59.0, 58.7, 54.2,49.4, 48.1, 46.7, 38.4, 32.8, 28.9, 25.1, 23.7, 22.8; APCIMS: m/z 544(Mþ 1); Anal. Calcd for C29H53Cl4N7O3 (687.3): C, 50.51; H, 7.75; N,14.22. Found: C, 50.66; H, 7.67; N, 14.23.

4.4. General method for the synthesis of D/L-lysyl/arginyl/ornithyl-N-{4-[(6-methoxy-2-substituted-quinolin-8-yl)amino]pentyl}-D/L-asparagine/glutamine (101e124)

4.4.1. Removal of Z groupTo a solution of protected conjugates (0.12 mmol) and 10% PdeC

(0.04 g) in glacial acetic acid (1 mL) and CH3OH (20 mL) wasbubbled a slow stream of H2 gas for 4 h. The catalyst filtered andsolvent removed to afford product.

4.4.2. Removal of t-Boc and tert-butyl ester groupsA solution of protected conjugates (0.7 mmol) in 4 N HCl

(10 mL) was stirred at ambient temperature for 5 h. Insome cases, a solution of protected derivatives and 33% HBrin AcOH (5 mL) was stirred for 30 min. The solvent wasevaporated under reduced pressure to afford product as hygro-scopic salt.

4.4.3. L-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HBr (101)

Yield: 92%; hygroscopic solid; IR (KBr): 3385, 1726, 1659 cm�1;1H NMR (CD3OD): d 8.86 (m, 2H), 7.93 (m, 1H), 7.08 (s, 1H), 6.95 (s,1H), 3.98 (m, 4H), 3.86 (t, 1H, J¼ 6.9 Hz), 3.31 (m, 1H), 2.99 (t, 2H,J¼ 7.3 Hz), 2.82 (t, 2H, J¼ 5.0 Hz), 1.98 (d, 2H, J¼ 6.6 Hz), 1.75 (m,10H), 1.37 (d, 3H, J¼ 6.2 Hz); 13C NMR (CD3OD): d 173.1, 172.0, 170.3,159.2, 145.7, 141.8, 133.9, 123.6, 98.3, 92.7, 67.2, 57.0, 54.3, 40.7, 38.2,28.4, 27.2, 22.9, 20.0; MALDIMS: m/z 503 (Mþ 1); Anal. Calcd forC25H41Br3N6O5 (742.0): C, 40.29; H, 5.54; N, 11.28. Found: C, 40.19;H, 5.61; N, 11.35.

4.4.4. L-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HCl (102)

Yield: 76%; hygroscopic solid; IR (KBr): 3433, 1730, 1658 cm�1;1H NMR (CD3OD): d 8.77 (m, 2H), 7.82 (m, 1H), 7.19 (s, 1H), 6.95 (s,1H), 3.94 (m, 4H), 3.82 (t, 1H, J¼ 6.3 Hz), 3.42 (m, 3H), 2.70 (t, 2H,J¼ 6.0 Hz), 1.88 (m, 2H), 1.63 (m, 8H), 1.27 (d, 3H, J¼ 5.7 Hz); 13CNMR (CD3OD): d 173.1, 172.0, 170.2, 162.9, 159.3, 159.0, 146.0, 141.5,140.3, 134.0, 123.6, 67.3, 57.0, 54.2, 45.1, 42.3, 38.2, 30.0, 27.3, 25.4,23.2, 20.1; MALDIMS: m/z 531 (Mþ 1); Anal. Calcd forC25H41Cl3N8O5 (638.2): C, 46.92; H, 6.46; N, 17.51. Found: C, 47.01;H, 6.53; N, 17.44.

4.4.5. L-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HBr (103)

Yield: 94%; hygroscopic solid; IR (KBr): 3400, 1630 cm�1; 1HNMR (CD3OD): d 8.88 (m, 2H), 7.94 (m, 1H), 7.12 (s, 1H), 6.97 (s, 1H),4.12 (m, 1H), 3.98 (s, 3H), 3.78 (m, 2H), 3.27 (t, 2H, J¼ 6.2 Hz), 3.07(t, 2H, J¼ 6.1 Hz), 2.84 (m, 2H), 1.98 (m, 8H), 1.37 (d, 3H, J¼ 6.2 Hz);13C NMR (CD3OD): d 172.8, 172.2, 170.9, 169.0, 161.7, 155.3, 144.8,140.8, 132.8, 122.6, 97.4, 91.2, 66.2, 56.0, 52.9, 49.5, 48.4, 47.8, 37.2,33.3, 28.9, 26.3, 23.2, 19.9; APCIMS: m/z 488.9 (Mþ 1); Anal. Calcdfor C24H39Br3N6O5 (728.0): C, 39.42; H, 5.38; N, 11.49. Found: C,39.59; H, 5.21; N, 11.65.

4.4.6. L-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HBr (104)

Yield: 91%; hygroscopic solid; IR (KBr): 3430, 1726, 1639 cm�1;1H NMR (CD3dOD): d 8.87 (m, 2H), 7.93 (m, 1H), 7.09 (s, 1H), 6.95 (s,1H), 4.12 (t, 1H, J¼ 6.3 Hz), 3.98 (s, 3H), 3.83 (m, 2H), 3.31 (t, 2H,J¼ 6.2 Hz), 3.02 (t, 2H, H¼ 6.0 Hz), 2.44 (t, 2H, J¼ 6.2 Hz), 2.23 (m,2H), 1.98 (m, 12H), 1.36 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD):d 176.5, 176.3, 173.3, 165.4, 148.3, 144.6, 142.6, 136.6, 126.3, 97.4,92.3, 59.7, 57.1, 56.4, 43.4. 43.3, 37.0, 36.2, 35.0, 31.4, 31.1, 29.9, 25.6,22.7; APCIMS: m/z 517 (Mþ 1); Anal. Calcd for C26H43Br3N6O5(756.0): C, 41.12; H, 5.71; N, 11.07. Found: C, 41.24; H, 5.86; N, 11.13.

4.4.7. L-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HCl (105)

Yield: 79%; hygroscopic solid; IR (KBr): 3435, 1729, 1630 cm�1;1H NMR (CD3OD): d 8.79 (m, 2H), 7.88 (m, 1H), 6.99 (s, 1H), 6.83 (s,1H), 4.04 (t, 1H, J¼ 5.3 Hz), 3.98 (s, 3H), 3.82 (m, 2H), 3.28 (m, 4H),2.82 (t, 2H, J¼ 5.7 Hz), 2.00 (m, 2H), 1.84 (m, 8H), 1.36 (d, 3H,J¼ 6.0 Hz); 13C NMR (CD3OD): d 173.9, 162.4, 158.8, 145.6, 135.3,133.8, 141.5, 123.2, 97.3, 91.4, 56.6, 54.0, 42.8, 41.9, 40.4, 37.9, 34.1,34.0, 29.6, 26.9, 25.0, 19.8; MALDIMS: m/z 545 (Mþ 1); Anal. Calcdfor C26H43Cl3N8O5 (652.2): C, 47.75; H, 6.63; N, 17.13. Found: C,47.84; H, 6.52; N, 17.27.

4.4.8. L-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HBr (106)

Yield: 88%; hygroscopic solid; IR (KBr): 3435, 1728, 1633 cm�1;1H NMR (CD3OD): d 8.75 (m, 2H), 7.84 (m, 1H), 7.00 (s, 1H), 6.86 (s,1H), 4.37 (t, 1H, J¼ 5.4 Hz), 4.05 (t, 1H, J¼ 6.1 Hz), 3.89 (s, 3H), 3.74(m, 1H), 3.22 (t, 2H, J¼ 5.9 Hz), 2.97 (t, 2H, J¼ 7.1 Hz), 2.35 (t, 2H,J¼ 7.4 Hz), 1.95 (m, 10H), 1.27 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD):d 173.2, 172.2, 171.9, 162.7, 145.5, 142.0, 139.9, 133.9, 130.9, 123.6,113.6, 96.4, 57.0, 53.9, 40.7, 38.1, 36.3, 34.3, 29.9, 27.3, 25.7, 24.2,19.9; APCIMS: m/z 503 (Mþ 1); Anal. Calcd for C25H41Br3N6O5(742.0): C, 40.29; H, 5.54; N, 11.28. Found: C, 40.17; H, 5.49; N, 11.39.

4.4.9. L-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HBr (107)

Yield: 84%; hygroscopic solid; IR (KBr): 3435, 1638 cm�1; 1HNMR (CD3OD): d 8.88 (m, 2H), 7.94 (m, 1H), 7.08 (s, 1H), 6.94 (s, 1H),4.05 (m,1H), 3.98 (s, 3H), 3.83 (m,1H, N-CH), 3.26 (t, 1H, J¼ 5.8 Hz),3.03 (m, 4H), 2.84 (m, 2H), 1.79 (m, 10H), 1.37 (d, 3H, J¼ 6.3 Hz); 13CNMR (CD3OD): d 173.1, 172.0, 170.3, 162.8, 145.8, 141.7, 140.0, 133.9,123.6, 98.1, 92.3, 67.2, 57.1, 48.6, 40.8, 38.2, 34.3, 32.3, 28.4, 27.3,22.9, 20.1; APCIMS:m/z 503 (Mþ 1); Anal. Calcd for C25H41Br3N6O5(742.0): C, 40.29; H, 5.54; N,11.28. Found: C, 40.35; H, 5.38; N,11.42.

4.4.10. D-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HBr (108)

Yield: 76%; hygroscopic solid; IR (KBr): 3421, 1721, 1665 cm�1;1H NMR (CD3OD): d 8.83 (m, 2H), 7.93 (m, 1H), 6.96 (s, 1H), 6.78 (s,1H), 4.01 (m, 4H), 3.83 (m, 2H), 3.31 (m, 4H, J¼ 5.4 Hz), 2.84 (m,2H), 1.80 (m, 8H), 1.37 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 172.2,168.1, 154.3, 145.8, 133.2, 123.9, 96.4, 91.8, 67.2, 57.5, 54.6, 49.5, 48.3,46.8, 41.5, 38.6, 32.3, 28.9, 26.3, 23.2, 21.1; APCIMS: m/z 531(Mþ 1); Anal. Calcd for C25H43Br3N8O5 (772.0): C, 38.73; H, 5.59; N,14.45. Found: C, 38.65; H, 5.73; N, 14.28.

4.4.11. D-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HBr (109)

Yield: 89%; hygroscopic solid; IR (KBr): 3435, 1637 cm�1; 1HNMR (CD3OD): d 8.85 (m, 2H), 7.92 (m, 1H), 7.08 (s, 1H), 6.96 (s, 1H),4.92 (m, 1H), 4.08 (s, 3H), 3.98 (m, 1H), 3.29 (t, 1H, J¼ 5.4 Hz), 3.02(m, 4H), 2.84 (m, 2H), 1.95 (m, 8H), 1.37 (d, 3H, J¼ 6.3 Hz); 13C NMR(CD3OD): d 173.2, 169.1, 155.3, 145.2, 133.9, 123.6, 97.4, 90.8, 66.2,

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241240

57.0, 53.9, 49.5, 48.4, 47.8, 41.5, 38.9, 34.3, 29.9, 27.3, 24.2, 19.9;APCIMS:m/z 489 (Mþ 1); Anal. Calcd for C24H39Br3N6O5 (728.0): C,39.42; H, 5.38; N, 11.49. Found: C, 39.37; H, 5.28; N, 11.57.

4.4.12. L-Lysyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HCl (110)

Yield: 85%; hygroscopic solid; IR (KBr): 3430, 1730, 1629 cm�1;1H NMR (CD3OD): d 8.86 (m, 2H), 7.91 (m, 1H), 7.18 (s, 1H), 6.91 (s,1H), 4.47 (t, 1H, J¼ 5.1 Hz), 4.04 (t, 1H, J¼ 5.7 Hz), 3.98 (s, 3H), 3.87(m, 1H), 3.25 (t, 2H, J¼ 5.6 Hz), 2.41 (t, 2H, J¼ 6.1 Hz), 1.96 (m, 4H),1.73 (m, 8H), 1.36 (d, 3H, J¼ 5.7 Hz); 13C NMR (CD3OD): d 173.9,172.2, 169.2, 140.1, 132.6, 122.1, 98.7, 96.1, 65.9, 39.3, 31.9, 30.9, 27.4,27.0, 25.7, 21.5; APCIMS: m/z 517 (Mþ 1); Anal. Calcd forC26H43Cl4N6O5 (659.2): C, 47.21; H, 6.55; N,12.71. Found: C, 47.14; H,6.48; N, 12.63.

4.4.13. D-Arginyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HCl (111)

Yield: 92%; hygroscopic solid; IR (KBr): 3408, 1645 cm�1; 1HNMR (CD3OD): d 8.83 (m, 2H), 7.91 (m, 1H), 7.17 (s, 1H), 6.91 (s, 1H),4.45 (t, 1H, J¼ 6.0 Hz), 3.98 (s, 3H), 3.92 (t, 1H, J¼ 6.8 Hz), 3.31 (m,5H), 2.42 (t, 2H, J¼ 5.3 Hz), 2.01 (m, 2H), 1.75 (m, 8H), 1.28 (d, 3H,J¼ 6.1 Hz); 13C NMR (CD3OD): d 175.6, 174.5, 170.5, 159.0, 145.9,142.7, 141.6, 134.0, 123.1, 107.6, 93.8, 67.3, 57.1, 54.3, 53.5, 45.5, 42.3,33.4, 30.0, 29.0, 27.1, 25.5, 23.1, 20.1; ESIMS: m/z 545 (Mþ 1); Anal.Calcd for C26H43Cl3N8O5 (652.2): C, 47.75; H, 6.63; N, 17.13. Found:C, 47.64; H, 6.54; N, 17.21.

4.4.14. D-Ornithyl-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HBr (112)

Yield: 92%; hygroscopic solid; IR (KBr): 3430, 1730, 1629 cm�1;1H NMR (CD3OD): d 8.79 (m, 2H), 7.85 (m, 1H), 6.99 (s, 1H), 6.85 (s,1H), 4.39 (t, 1H, J¼ 7.1 Hz), 4.09 (t, 1H, J¼ 5.4 Hz), 3.89 (s, 3H), 3.74(m, 1H), 3.22 (t, 2H, J¼ 5.9 Hz), 2.98 (t, 2H, J¼ 7.1 Hz), 2.37 (t, 2H,J¼ 7.3 Hz), 2.14 (m, 2H), 1.80 (m, 8H), 1.27 (d, 3H, J¼ 6.3 Hz); 13CNMR (CD3OD): d 175.2, 173.8, 173.6, 170.2, 162.7, 145.7, 141.8, 140.0,133.9, 123.6, 67.3, 57.1, 54.0, 45.1, 40.7, 34.3, 33.5, 29.9, 28.7, 27.2,24.3, 22.9, 20.9; APCIMS: m/z 503 (Mþ 1); Anal. Calcd forC25H41Br3N6O5 (742.0): C, 40.29; H, 5.54; N, 11.28. Found: C, 40.24;H, 5.42; N, 11.16.

4.4.15. L-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HBr (113)

Yield: 93%; hygroscopic solid; IR (KBr): 3435, 1635 cm�1; 1HNMR (CD3OD): d 8.35 (d, 1H, J¼ 8.7 Hz), 7.82 (d, 1H, J¼ 8.7 Hz), 7.57(s, 1H), 7.48 (s, 1H), 4.09 (m, 4H), 3.31 (m, 2H), 3.01 (t, 2H,J¼ 5.7 Hz), 2.79 (t, 2H, J¼ 6.4 Hz), 1.96 (m, 2H, J¼ 6.1 Hz), 1.78 (m,10H), 1.50 (s, 9H), 1.41 (d, 3H, J¼ 6.5 Hz); 13C NMR (CD3OD): d 173.1,171.9, 162.1, 156.7, 145.6, 143.8, 138.1, 122.4, 97.6, 91.3, 67.2, 57.1,54.3, 40.7, 40.4, 39.7, 38.2, 32.3, 30.8, 28.4, 26.9, 23.2, 22.9, 21.4;MALDIMS:m/z 559 (Mþ 1); Anal. Calcd for C29H49Br3N6O5 (798.1):C, 43.46; H, 6.16; N, 10.49. Found: C, 43.56; H, 6.25; N, 10.58.

4.4.16. L-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HCl (114)

Yield: 79%; hygroscopic solid; IR (KBr): 3445, 1622 cm�1; 1HNMR (CD3OD): d 8.75 (d, 1H, J¼ 8.4 Hz), 8.50 (d, 1H, J¼ 8.4 Hz), 7.85(m, 2H), 4.01 (t, 1H, J¼ 7.3 Hz), 3.88 (m, 4H), 3.57 (m, 1H), 3.21 (m,2H), 2.76 (t, 2H, J¼ 5.8 Hz), 2.67 (d, 2H, J¼ 7.3 Hz), 1.97 (m, 8H), 1.49(s, 9H), 1.38 (d, 3H, J¼ 6.2 Hz); 13C NMR (CD3OD): d 172.1, 169.7,159.3, 146.4, 144.9, 135.6, 129.0, 118.6, 97.5, 91.3, 67.3, 62.5, 57.8,54.4, 45.0, 42.2, 39.6, 30.7, 25.3, 23.2, 21.8; APCIMS: m/z 587(Mþ 1); Anal. Calcd for C29H49Cl3N8O5 (694.2): C, 50.04; H, 7.10; N,16.10. Found: C, 50.10; H, 7.23; N, 16.05.

4.4.17. L-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-asparagine$3HBr (115)

Yield: 96%; hygroscopic solid; IR (KBr): 3425, 1658 cm�1; 1HNMR (CD3OD): d 8.37 (d, 1H, J¼ 8.7 Hz), 7.82 (d, 1H, J¼ 8.7 Hz), 7.61(s, 1H), 7.49 (s, 1H), 4.11 (m, 1H), 3.99 (s, 3H), 3.73 (t, 1H, J¼ 5.7 Hz),3.35 (m, 1H), 3.22 (t, 2H, J¼ 6.0 Hz), 3.05 (t, 2H, J¼ 5.7 Hz), 2.82 (m,2H), 2.02 (m, 8H), 1.50 (s, 9H), 1.42 (d, 3H, J¼ 6.5 Hz); 13C NMR(CD3OD): d 172.4, 172.1, 170.8, 159.4, 156.2, 144.9, 144.2, 135.3, 134.7,129.9, 121.8, 96.7, 91.6, 55.2, 54.7, 52.3, 47.8, 39.8, 39.5, 34.0, 32.4,31.8, 29.6, 28.4, 28.2, 27.9, 26.2, 22.4, 20.5; APCIMS: m/z 545(Mþ 1); Anal. Calcd for C28H47Br3N6O5 (784.1): C, 42.71; H, 6.02; N,10.67. Found: C, 42.65; H, 6.20; N, 10.54.

4.4.18. L-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HCl (116)

Yield: 91%; hygroscopic solid; IR (KBr): 3421, 1712, 1667 cm�1;1H NMR (CD3OD): d 8.35 (d, 1H, J¼ 8.7 Hz), 7.81 (d, 1H, J¼ 8.7 Hz),7.56 (s,1H), 7.47 (s,1H), 4.47 (t, 1H, J¼ 6.4 Hz), 4.02 (m, 4H), 3.31 (m,1H), 3.23 (t, 2H, J¼ 6.5 Hz), 3.00 (t, 2H, J¼ 7.3 Hz), 2.38 (t, 2H,J¼ 6.8 Hz), 1.97 (m, 2H), 1.75 (m, 10H), 1.50 (s, 9H), 1.39 (d, 3H,J¼ 6.5 Hz); 13C NMR (CD3OD): d 170.6, 169.6, 161.3, 146.1, 144.5,135.6, 122.3, 98.4, 92.3, 67.3, 57.1, 54.3, 40.7, 40.3, 32.3, 30.8, 28.7,28.4, 26.9, 22.9, 21.3; APCIMS: m/z 573 (Mþ 1); Anal. Calcd forC30H51Cl3N6O5 (680.3): C, 52.82; H, 7.54; N, 12.32. Found: C, 52.88;H, 7.61; N, 12.41.

4.4.19. L-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HCl (117)

Yield: 86%; hygroscopic solid; IR (KBr): 3398, 1731, 1683 cm�1;1H NMR (CD3OD): d 8.74 (d, 1H, J¼ 8.5 Hz), 8.36 (d, 1H, J¼ 8.5 Hz),7.85 (m, 2H), 4.45 (t, 1H, J¼ 6.8 Hz), 4.05 (t, 1H, J¼ 5.4 Hz), 3.99 (s,3H), 3.34 (m, 5H), 2.37 (t, 2H, J¼ 6.1 Hz), 2.23 (m, 2H), 1.97 (m, 8H),1.50 (s, 9H),1.33 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 173.5,170.5,169.6, 159.2, 159.0, 155.4, 136.7, 135.5,131.2, 129.0, 126.0, 121.9, 98.4,67.3, 62.5, 57.9, 54.2, 45.4, 42.2, 40.4, 30.8, 30.7, 28.7, 26.9, 23.1, 21.7;APCIMS:m/z 601 (Mþ 1); Anal. Calcd for C30H51Cl3N8O5 (708.3): C,50.74; H, 7.24; N, 15.78. Found: C, 50.81; H, 7.32; N, 15.69.

4.4.20. L-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-D-glutamine$3HCl (118)

Yield: 95%; hygroscopic solid; IR (KBr): 3392, 3078, 1729, 1677,1641 cm�1; 1H NMR (CD3OD): d 8.37 (d, 1H, J¼ 8.4 Hz), 7.83 (d, 1H,J¼ 8.4 Hz), 7.62 (s, 1H), 7.49 (s, 1H), 4.47 (t, 1H, J¼ 6.3 Hz), 4.10 (t,1H, J¼ 6.9 Hz), 4.00 (s, 3H), 3.32 (m,1H), 3.24 (t, 2H, J¼ 5.4 Hz), 3.04(t, 2H, J¼ 6.0 Hz), 2.41 (t, 2H, J¼ 7.1 Hz), 2.01e1.76 (m, 10H), 1.51 (s,9H), 1.41 (d, 3H, J¼ 5.9 Hz); 13C NMR (CD3OD): d 173.8, 172.9, 168.6,156.7, 136.5, 134.9, 130.9, 128.6, 120.6, 117.0, 93.4, 65.6, 58.8, 55.5,52.0, 38.8, 30.6, 29.1, 28.2, 25.2, 22.5, 21.4; APCIMS: m/z 559(Mþ 1); Anal. Calcd for C29H49Cl3N6O5 (666.2): C, 52.13; H, 7.39; N,12.58. Found: C, 52.25; H, 7.44; N, 12.45.

4.4.21. L-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HCl (119)

Yield: 81%; hygroscopic solid; IR (KBr): 3415, 1710, 1672 cm�1;1H NMR (CD3OD): d 8.25 (d, 1H, J¼ 8.5 Hz), 7.73 (d, 1H,J¼ 8.5 Hz), 7.45 (s, 1H), 7.39 (s, 1H), 4.68 (t, 1H, J¼ 6.1 Hz), 3.99 (s,3H), 3.82 (t, 1H, J¼ 5.8 Hz), 3.35 (m, 3H), 3.00 (t, 2H, J¼ 5.6 Hz),2.73 (d, 2H, J¼ 6.1 Hz), 1.98 (m, 10H), 1.51 (s, 9H), 1.38 (d, 3H,J¼ 6.3 Hz); 13C NMR (CD3OD): d 172.4, 170.5, 168.6, 166.7, 157.1,155.0, 148.9, 138.3, 136.0, 134.5, 133.9, 130.1, 128.2, 119.6, 96.3,56.0, 55.5, 43.3, 41.3, 39.0, 38.7, 37.6, 36.9, 35.8, 34.1, 31.3, 30.7,29.2, 28.7, 26.5, 22.2; APCIMS: m/z 559 (Mþ 1); Anal. Calcd forC29H49Cl3N6O5 (666.2): C, 52.13; H, 7.39; N, 12.58. Found: C,52.24; H, 7.46; N, 12.51.

K. Kaur et al. / European Journal of Medicinal Chemistry 52 (2012) 230e241 241

4.4.22. D-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HCl (120)

Yield: 77%; hygroscopic solid; IR (KBr): 3415, 1723, 1648 cm�1;1H NMR (CD3OD): d 8.35 (d, 1H, J¼ 8.8 Hz), 7.89 (d, 1H, J¼ 8.8 Hz),7.55 (m, 2H), 4.43 (m, 1H), 4.02 (t, 1H, J¼ 6.7 Hz), 3.99 (s, 3H), 3.52(m, 1H), 3.31 (m, 2H), 2.82 (t, 2H, J¼ 5.8 Hz), 2.67 (m, 2H), 1.96 (m,8H), 1.49 (s, 9H), 1.38 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 172.7,171.6, 169.2, 159.2, 158.7, 155.1, 136.3, 135.2, 130.9, 128.6, 120.5, 97.4,92.1, 60.6, 57.6, 53.8, 44.7, 41.9, 40.0, 39.4, 39.3, 38.2, 31.9, 30.5, 29.6,28.1, 26.5, 25.3, 23.0, 22.1, 20.9; APCIMS: m/z 587 (Mþ 1); Anal.Calcd for C29H49Cl3N8O5 (694.2): C, 50.04; H, 7.10; N, 16.10. Found:C, 50.14; H, 7.15; N, 16.18.

4.4.23. D-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-asparagine$3HCl (121)

Yield: 98%; hygroscopic solid; IR (KBr): 3397,1725,1661 cm�1; 1HNMR (CD3OD): d 8.39 (d, 1H, J¼ 8.3 Hz), 7.82 (d, 1H, J¼ 8.3 Hz), 7.66(s,1H), 7.49 (s,1H), 4.87 (t,1H, J¼ 5.7 Hz), 4.12 (t,1H, J¼ 6.8 Hz), 4.00(s, 3H), 3.56 (m, 1H), 3.24 (m, 4H), 2.83 (d, 2H, J¼ 5.7 Hz), 2.06 (m,8H), 1.51 (s, 9H), 1.35 (d, 3H, J¼ 6.3 Hz); 13C NMR (CD3OD): d 176.3,171.4, 168.7, 156.6, 136.5, 134.8, 131.0, 128.5, 120.5, 116.8, 58.6, 55.5,43.3, 42.3, 38.9, 38.0, 36.5, 35.7, 34.6, 30.6, 29.1, 28.1, 25.1, 23.4, 22.6,21.9; APCIMS: m/z 545 (Mþ 1); Anal. Calcd for C28H47Cl3N6O5

(652.2): C, 51.42; H, 7.24; N, 12.85. Found: C, 51.36; H, 7.32; N, 12.93.

4.4.24. L-Lysyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HCl (122)

Yield: 83%;hygroscopic solid; IR (KBr): 3409,1724,1663 cm�1; 1HNMR (CD3OD): d 8.25 (d, 1H, J¼ 8.7 Hz), 7.71 (d, 1H, J¼ 8.7 Hz), 7.51(s, 1H), 7.43 (s, 1H), 4.38 (m, 1H), 3.95 (t, 1H, J¼ 6.2 Hz), 3.89 (s, 3H),3.64 (m,1H), 3.12 (t, 2H, J¼ 6.6 Hz), 2.90 (t, 2H, J¼ 7.5 Hz), 2.26 (t, 2H,J¼ 7.1 Hz),1.90 (m,12H),1.40 (s, 9H),1.29 (d, 3H, J¼ 6.5 Hz); 13CNMR(CD3OD): d 173.2, 171.9, 161.3, 156.8, 146.4, 144.3, 135.3, 128.5, 120.5,98.6, 91.7, 55.4, 52.5, 38.9, 38.5, 37.9, 31.6, 30.5, 29.0, 26.9, 26.6, 25.2,21.9, 21.1; APCIMS: m/z 573 (Mþ 1); Anal. Calcd for C30H51Cl3N6O5(680.3): C, 52.82; H, 7.54; N,12.32. Found: C, 52.93; H, 7.47; N,12.26.

4.4.25. D-Arginyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HCl (123)

Yield: 78%; hygroscopic solid; IR (KBr): 3435, 1726, 1662 cm�1;1H NMR (CD3OD): d 8.45 (d, 1H, J¼ 8.3 Hz), 7.81 (d, 1H, J¼ 8.3 Hz),7.17 (m, 2H), 4.46 (m, 1H), 4.05 (t, 1H, J¼ 6.3 Hz), 3.99 (s, 3H), 3.85(m, 1H), 3.34 (m, 4H), 2.36 (t, 2H, J¼ 7.1 Hz), 1.97 (m, 10H), 1.50 (s,9H), 1.38 (d, 3H, J¼ 6.5 Hz); 13C NMR (CD3OD): d 172.1, 168.1, 161.3,157.2, 144.9, 143.7, 136.1, 122.4, 98.1, 92.3, 65.5, 52.5, 42.9, 40.5, 29.0,28.3, 21.6; APCIMS: m/z 601 (Mþ 1); Anal. Calcd for C30H51Cl3N8O5(708.3): C, 50.74; H, 7.24; N, 15.78. Found: C, 50.82; H, 7.15; N, 15.89.

4.4.26. D-Ornithyl-N-{4-[(2-tert-butyl-6-methoxyquinolin-8-yl)amino]pentyl}-L-glutamine$3HCl (124)

Yield: 95%; hygroscopic solid; IR (KBr): 3390, 1728, 1663 cm�1;1H NMR (CD3OD): d 8.34 (d, 1H, J¼ 8.5 Hz), 7.81 (d, 1H, J¼ 8.5 Hz),7.69 (s, 1H), 7.46 (s, 1H), 4.46 (m, 1H), 4.08 (m, 4H), 3.22 (m, 3H),3.02 (t, 2H, J¼ 6.4 Hz), 2.36 (t, 2H, J¼ 6.9 Hz), 1.99 (m, 10H), 1.51 (s,9H), 1.30 (d, 3H, J¼ 6.2 Hz); 13C NMR (CD3OD): d 173.4, 172.9, 163.4,144.1, 143.8, 133.6, 120.5, 118.1, 95.4, 65.4, 55.3, 43.2, 38.7, 38.4, 37.9,31.8, 30.5, 28.9, 28.0, 27.1, 25.1, 22.4, 21.7; APCIMS:m/z 559 (Mþ 1);Anal. Calcd for C29H49Cl3N6O5 (666.2): C, 52.13; H, 7.39; N, 12.58.Found: C, 52.20; H, 7.26; N, 12.52.

Acknowledgements

Kirandeep Kaur thanks the Council of Scientific and IndustrialResearch (CSIR), New Delhi for the award of Senior ResearchFellowship. The antimicrobial testing was supported by the NIH,

NIAID, Division of AIDS, grant no. AI 27094. Support from the USDAAgricultural Research Service Specific Cooperative Agreement No.58-6408-2-0009 is also acknowledged in the in vitro screening ofantimicrobial, antiprotozoal, and cytotoxic activity at NCNPR,University of Mississippi.

Appendix 1. Supplementary material

Supplementary material associated with this article can befound, in the online version, at doi:10.1016/j.ejmech.2012.03.019.

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