Non-classical antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines and 2,4-Diamino-6(5H)-oxopyrimidines, synthesis and antitumor studies

Bioorganic & Medicinal Chemistry 11 (2003) 145–157

Non-classical Antifolates, 5-(N-Phenylpyrrolidin-3-yl)-2,4,6-

triaminopyrimidines and 2,4-Diamino-6(5H)-oxopyrimidines,

Synthesis and Antitumor Studies

Yen-Lin Huang,^a,bChyun-Feng Lin,^a,bYi-Jen Lee,^aWei-Wei Li,^cTing-Chou Chao,^c

Valeriy A. Bacherikov,^aKuo-Tung Chen,^bChin-Ming Chen^bandTsann-Long Su ^a,*

^aLaboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

^bDepartment of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan

^cMolecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

Received5 December 2001; accepted26 April 2002

Abstract—A series of non-classical antifolates, namely 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines (25a–i) and2,4-dia-

mino-(N-phenylpyrrolidin-3-yl)-6(5H)-oxopyrimidines (26a,b,c,f,h,i) was synthesizedandevaluatedfor their in vitro cytotoxicity.

Reacting aniline derivatives with 1,4-dibromo-2-butanol gave 1-phenyl-3-pyrrolidinols (19a-–i), which were oxidized to pyrrolidin-

3-ones (20a–i). The Knoevenagel reaction of 20a–i with malononitrile or ethyl cyanoacetate gave 3-(dicyanomethylene)- (21a–i) and

3-[cyano(ethoxycarbonyl)methylene]-pyrrolidines (22a,b,c,f,h,i), respectively, which were subsequently reduced to the corresponding

3-(dicyano)methyl- or 3-[cyano(ethoxycarbonyl)methyl)]pyrrolidines (23a–i and 24a,b,c,f,h,i, respectively). Condensation of either

23a–i or 24a,b,c,f,h,i with guanidine aﬀorded the target compounds. The cytotoxicity of these compounds was evaluated based on

their ability to inhibit various human tumors (human colon adenocarcinoma COLO 205, lung carcinoma H23 and its adriamycin

resistant cell line H23/0.3, T-cell leukemia MOLT-4, promyelocytic leukemia HL-60, andT-cell acute lymphocytic leukemia CCRF-

CEM) cell growth in culture. These studies revealed that the 2,4,6-triaminopyrimidine derivatives were more cytotoxic than the 2,4-

diamino-6(5H)-oxopyrimidine counter parts, in which the latter was inactive in all testing systems. The 2,4,6-triaminopyrimidine

derivatives bearing halogen substituent on the phenyl ring (25f,h,i) were cytotoxic in all culturedleukemia cell growth. Among these

compounds, 5-(4-ﬂuoro and 4-chlorophenyl)-2,4,6-triaminopyrimidines (25e and 25h, respectively) were more potent than metho-

trexate (MTX) in inhibiting of H23/0.3 cell growth. These compounds inhibit the folate metabolic pathways as indicated by tritium

release from [5-³H]deoxyuridine in MTX sensitive human ﬁbrosarcoma HT-1080 cells. Dihydrofolate reductase is the major target

for 25f,h,i, as shown by leucovorin (LV) rescue of MTX cytotoxicity.

Introduction

subject in the development of chemotherapeutic agents.³

Unfortunately, several problems are associatedwith the

clinical use of MTX such as: (1) high levels of toxicity to

Research andedvelopment of antifolates for cancer

chemotherapy have been one of a major focus in anti-

cancer drug design. The discovery of aminopterin (AP)

andmethotrexate (MTX) having a potent antitumor

eﬃcacy in cancer patients,¹leadto intensive investiga-

tions of numerous classical antifolates carrying a p-

4

bone marrow andgastrointestinal mucosa, (2) resis-

tance to the drug accompanied by an elevation of levels

of the target enzyme, DHFR,⁵and(3) the decrease in

the transport of the drug.^6,7The most common reason

of intrinsic resistance is a defect in the membrane

transport of MTX.^8,9To minimize problems causedby

MTX, various antifolates with modiﬁcations in the

pteridine ring (such as deazaaminopterins), phenyl moi-

ety and their tetrahydrofolate analogues were studied.¹⁰

The openedB-ring compounds, such as N-[4-(2,4-dia-

mino-6-oxo-1H,6H-pyrimidin-5-yl)butyl]benzoyl]-l-glu-

tamic acidandits 2,4,6-triamino derivatives ( 1^11,12and

2,^13,14respectively, Fig. 1) are synthesizedfor both in

2

aminobenzoyl-l-glutamic acidmoiety. The inhibition

of two important enzymes, dihydrofolate reductase

(DHFR) andthymidylate synthase (TS, both involved

in the metabolic pathway of folic acid), is the main

*Corresponding author. Tel.: +886-2-2782-7685; fax: +886-2-2782-

7685; e-mail: tlsu@ibms.sinica.edu.tw

PII: S0968-0896(02)00238-9

146

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

Figure 1. Structures of some antifolates.

vitro andin vivo antitumor studies. ¹⁵Compounds 1 and 2

are viewedas 7-desmethylene analogues of the potent

purine biosynthesis inhibitors, DDATHF (3)¹⁶and

DATHF (4).¹⁷Rosowsky et al.¹⁸synthesized N-[4-[3-(2,4-

diamino-6(5H)-oxopymidin-5-yl)pyrrolidino]benzoyl]-l-

glutamic acid( 5) andits 1,2,6-triamino derivatives ( 6),

which are 5-deaza-7-desmethylene analogues of 5,10-

methylene-5,6,7,8-tetrahydrofolic acid (7). Compound 5

and 6 are goodsubstrates for mouse liver folylpolyglu-

tamate synthetase andthe latter is a DHFR inhibitor,

which inhibits the growth of culture tumor cells (SCC25

human squamous cell carcinoma).¹⁸

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

147

Numerous antifolates, not carrying the glutamate moiety

(i.e., non-classical antifolates), have versatile biological

activity. The non-classical antifolates are developed in

an attempt to overcome the resistance causedby MTX.

These compounds are envisioned to be more lipophilic,

thus can enter into the cell via passive diﬀusion. Com-

pounds belonging to non-classical antifolates are deri-

vatives of pyrido[2,3-d]pyrimidine^19ꢀ22[such as

piritrexim (PTX, 8)¹⁹andtrimetrexate (TMQ, 9)²²],

quinazoline,^23,24andpyrimidine [such as trimethoprim

(TMP, 10),²⁵pyrimethamine (PYR, 11)²⁶andmetoprine

(DDMP, 12).²⁶Although reports demonstrated TMP as

a poor inhibitor of mammalian DHFR, it is approxi-

mately 10-foldmore potent against Pneumocystis carri-

nii DHFR.²⁷Similarly, PYR analogues with poor

potency against P. carrinii DHFR does not show sig-

niﬁcant selectivity.²⁷Unlike TMP andPYR, both ( 8)

and( 9) inhibit mammalian DHFR more potently than

pathogen DHFRs. However, they do not oﬀer any

beneﬁcial selectivity.²⁸Therefore, many of these agents

have been developed as potent antimicrobial and/or

anticancer activities.^29ꢀ32For example, PTX andTMQ

are in clinical trials for treatment of AIDS-related

opportunistic infections^33,34as well as advanced bladder

cancer.^35,36Studies also show DDMP to be a potentially

useful drug against brain tumors.³⁷

ﬁndings show that the halogen substituted derivatives of

2,4,6-triaminopyrimidine are cytotoxic to the cell

growth of several of human leukemias in culture. These

compounds inhibit the folate metabolic pathways as

indicated by tritium release from [5-³H]-deoxyuridine in

MTX sensitive human ﬁbrosarcoma HT-1080 cells.

These studies have also revealed dihydrofolate reductase

as the major target for 25f,h,i, as shown by leucovorin

(LV) rescue of MTX cytotoxicity.

Chemistry

The synthetic routes for the preparation of our com-

pounds are based on Rosowsky’s publication¹⁸andour

previous report³⁷with some modiﬁcations. In our pre-

vious study,³⁷we synthesized1-(4-alkyloxycarbonyl-

phenyl)-3-pyrrolidinone by the condensation of 1,4-

dibromo-2-butanol (17) with alkyl p-aminobenzoate,

followed by the oxidation (DCC/pyridine/DMSO/TFA)

of the product 1-(4-alkyloxycarbonylphenyl)-3-pyrroli-

dinol. Using the same procedure, treating 17 with a

variety of substitutedanilines ( 18a–i) in triethyl phos-

phate in the presence of potassium carbonate under

reﬂux for 4–6 h aﬀorded 1-phenyl-3-pyrrolidinols (19a–

i) in low to moderate yields (16–62%), depending on the

substituent in the phenyl ring (Scheme 1). Condensation

of 17 with aniline (18a), 2-Cl-, 4-Cl-, or 4-Br-aniline

(18g, 18h, or 18i, respectively) aﬀorded 1-phenyl-3-pyr-

rolidinols (19a,g,h,i, respectively) in moderate yield,

while reacting 17 with ﬂuoro-substitutedanilines ( 18e,f),

anilines bearing a electron-withdrawing function (i.e.,

CN andNO ₂, 18c,d) or a electron-donating function

(OMe, 18b) gave a low yieldof 1-phenyl-3-pyrrolidinols

(19b–d). The original synthetic methodfor 1-phenyl-3-

pyrrolidinone was reported by Taylor et al.,³⁸who syn-

thesized1-[(4- tert-butoxycarbonyl)phenyl]-3-pyrrolidi-

none by the condensation of 3-pyrrolidinol with tert-

butyl 4-ﬂuorobenzoate, followedby oxiadtion. Our

methodology for the synthesis of 1-phenyl-3-pyrrolidi-

nols 19 has some advantage since various N-substituted

3-pyrrolidinols can be prepared under the facile reaction

Previously, we synthesized1,3-iadminopyrrolo[3

⁰,4⁰:

4,5]pyrido[2,3-d]pyrimidine derivatives (13 and 14, Fig.

1). Compound 13 is an analogue of 5,10-methylene-

FAH₄^36,37andis 1/10 as potent as MTX against human

leukemia Hl-60 cell in culture. Compound 14 lacks the

p-aminobenzoyl-l-glutamic acidmoiety andis an intri-

cate subject for biological evaluations because of its

poor solubility. To continue our research on the devel-

opment of non-classical antifolates as potential anti-

tumor agents, we synthesizeda series of 5-( N-

phenylpyrrolidin-3-yl)-2,4,6-triamniopyrimidines (15) and

2,4-diamino-5-(N-phenylpyrrolidin-3-yl)-6(5H)-oxo-

pyrimidine (16) derivatives, which can be considered as

5-deaza-7-desmethylene analogues of 14 as well as deri-

vatives of 5 and 6 without the glutamate moiety. Our

Scheme 1. Synthesis of non-classical antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines and 2,4-diamino-6(5H)-oxopyrimidines:

(a) R=H; (b) R=3-OMe; (c) R=4-CN; (d) R=4-NO₂; (e) R=2-F; (f) R=4-F; (g) R=2-Cl; (h) R=4-Cl; (i) R=4-Br. Reagents andconditions: (i)

(EtO)₃PO/K₂CO₃/150 ^ꢁC; (ii) DCC/DMSO/pyridine/TFA, 0 ^ꢁC; (iii) CH₂(CN)₂or NCCH₂COOEt/DBU/benzene/0 ^ꢁC to rt; (iv) NaBH₃CN/AcOH;

(v) guanidine dicarbonate/K₂CO₃/DMF.

148

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

conditions by the reaction of substituted anilines with

inexpensive 17.

compounds are shown in Table 2. Generally, the pro-

tons at C-4 appearedin higher ﬁeldin the area of

1.88–2.60, while the protons at C-2 andC-5 appearedin

the region of d 3.0–3.75 andC-3 protons are showedat

d

Using the Pﬁtzner–Moﬀatt³⁹procedure, 3-pyrrolidinols

19a–i were oxidized to give pyrrolidinones 20a–i in

moderate to good yield. The Knoevenagel reaction of

pyrrolidin-3-ones 20a–i with malononitrile or ethyl

cyanoacetate aﬀorded 3-(dicyanomethylene)pyrrolidines

(21a–i) or 3-[cyano(ethoxycarbonyl)methylene]pyrroli-

dines (22a,b,c,f,h,i), respectively. In most cases, the

reactions were carriedout in dry ether in the presence of

base catalyst (DBU) andthe driedmolecular sieves at

0 ^ꢁC, andthe reaction mixture was then graudally

warmedup to room temperature andcontinuously stir-

redfor 2–8 h. Compounds 21e and 21g were prepared

by reacting 20e and 20g, respectively, with malononitrile

in dry ethanol in the presence of KF. Products

21a,b,f,h,i precipitatedduring the course of reaction and

were isolated in a stable solid form. The crude products

21c,d,e,g did not precipitate out and contained a small

amount of the starting material along with by-products

andwere usedin the next reaction. Since the desired

product and the starting ketone have the same R_fvalue

on the TLC (SiO₂) with a variety of solvent system, we

were unsuccessful in isolating the product by column

chromatography. In these cases, prolongation of the

reaction time or addition of excess malononitrile usually

resultedmore edcomposition. Similarly, reacting 3-

pyrrolidinones 20a,b,c,f,h,i with ethyl cyanoacetate

using N,N-dimethylaminoethanol as base aﬀorded

22a,b,c,f,h,i in moderate to good yield. The oleﬁn func-

tion of 21a–i and 22a,b,c,f,h,i was reduced by sodium

cyanoborohydride in acetic acid to give 3-(dicyano-

methyl)pyrrolidines (23a–i) and3-[cyano(ethoxy-

carbonyl)methyl]pyrrolidines (20a,b,c,f,h,i), respectively.

Both were then condensed with guanidine dicarbonate

d 2.95–4.40. The chemical shifts of these protons are

aﬀectedby the substituent at C-3, especially for those

compounds with pyrimidine moiety at C-3.

CHN analysis of newly synthesized compounds

CompdFormula

Anal. calcd

Found

H

(%) (%) (%) (%) (%) (%)

C

H

N

C

N

19a

19b

19c

19d

19e

19f

19g

19h

C₁₀H₁₃NO

73.59 8.03 8.58 73.60 8.05 8.52

68.37 7.82 7.25 68.21 7.75 7.32

70.19 6.43 14.88 70.16 6.45 14.84

57.68 5.81 13.45 57.55 5.76 13.24

66.28 6.67 7.73 66.12 6.50 7.61

66.28 6.67 7.73 65.98 6.72 7.66

C₁₁H₁₅NO₂:

C₁₁H₁₂N₂O

C₁₀H₁₂N₂O₃

C₁₀H₁₂FNO

C₁₀H₁₂ClNO 60.76 6.12 7.09 60.58 6.05 7.15

.

C₁₀H₁₂ClNO 57.49 6.39 6.70 57.34 6.31 6.22

5/8H₂O

C₁₀H₁₂BrNO 49.61 5.00 33.01 49.75 5.10 33.10

19i

20a

20b

20c

C₁₀H₁₁NO

C₁₁H₁₃NO₂

C₁₁H₁₀N₂O

1/5H₂O

74.51 6.88 8.69 74.71 6.72 8.58

69.09 6.85 7.33 69.00 7.10 7.02

6.95 5.52 14.76 7.02 5.31 14.56

.

20d

20e

20f

20g

20h

20i

C₁₀H₁₀N₂O₃

C₁₀H₁₀FNO

58.25 4.89 13.59 58.50 4.79 13.24

67.03 5.63 7.82 66.92 5.45 7.67

67.03 5.63 7.82 66.87 5.65 7.77

C₁₀H₁₀ClNO 61.39 5.15 7.16 61.21 5.06 7.22

C₁₀H₁₀ClNO 61.39 5.15 7.16 61.53 5.13 7.00

C₁₀H₁₀BrNO 50.02 4.20 5.83 49.88 4.23 5.82

21a

21b

21f

21h

21i

22a

22b

22c

22f

22h

22i

C₁₃H₁₁N₃

74.62 5.30 20.08 74.56 5.54 19.90

70.27 5.48 17.56 70.10 5.42 17.38

68.71 4.44 18.49 68.61 4.68 18.33

64.64 4.14 17.24 64.81 4.36 17.56

54.19 3.50 14.58 54.02 3.65 14.35

70.29 6.29 10.93 70.10 6.16 10.79

71.11 6.71 10.36 71.02 6.65 10.22

68.31 5.37 14.94 68.21 5.19 14.85

andfurnishedthe desiredracemic 5-(

N-phenylpyrroli-

C₁₄H₁₃N₃O

C₁₃H₁₀FN₃

C₁₃H₁₀ClN₃

C₁₃H₁₀BrN₃

C₁₅H₁₆N₂O₂

C₁₆H₁₈N₂O₂

C₁₆H₁₅N₃O₂

din-3-yl)-2,4,6-triaminopyrimidines (25a–i) and2,4-di-

amino-(N-phenylpyrrolidin-3-yl)-6(5H)-oxopyrimidines

(26a,b,c,f,h,i), respectively, in a low to moderate yield. It

shouldbe noted, that our preliminary results revealed

that 2,4-diamino-(N-phenylpyrrolidin-3-yl)-6(5H)-oxo-

pyrimidines (26a,b,c,f,h,i) were inactive in inhibiting

various tumor cell growth in culture (see below) and

compounds 20d,e,g were diﬃcult to handle because of

their instability, therefore, we did not prepare 22d,e,g

for the synthesis of 26d,e,g.

C₁₅H₁₅FN₂O₂65.68 5.51 10.21 65.51 5.58 10.33

C₁₅H₁₅ClN₂O₂61.96 5.20 9.64 61.77 5.46 9.63

C₁₅H₁₅BrN₂O₂53.75 4.51 8.36 53.67 4.52 8.23

.

23a

C₁₃H₁₃N₃

1/10H₂O

C₁₄H₁₅N₃O

73.28 6.24 19.72 73.48 6.27 19.80

23b

23c

69.69 6.27 17.42 69.53 6.18 17.33

70.10 5.21 23.36 71.24 5.27 23.18

The structure of all compounds were determined and

conﬁrmedby IR and ¹H NMR spectroscopy methods

(Tables 1 and2, respectively). The ¹H NMR spectra of

3-substituted-N-phenylpyrrolidine derivatives reported

previously¹⁸were described without any assignments of

the chemical shift of each proton on the pyrrolidine

ring. Protons on the pyrrolidine ring appeared as multi-

spin systems andwere idﬃcult to assign from one-

dimensional spectra because of overlapping complex-

ities. In the present study we have analyzed the ¹H

NMR andCOSY spectra of the newly synthesized

compounds and assigned the chemical shifts of protons

of pyrrolidine ring, where orientation of substituents is

more important in a stereochemical point of view. The

parameters of ¹H NMR spectra of the synthesized

.

C₁₄H₁₂N₄

1/5H₂O

23d

23e

23f

23g

C₁₃H₁₂N₄O₂

C₁₃H₁₂FN₃

60.93 4.72 21.86 60.81 4.65 21.71

68.11 5.28 18.33 67.95 5.44 18.66

68.11 5.28 18.33 68.09 5.34 18.46

48.53 4.87 13.06 48.45 4.68 12.83

.

C₁₃H₁₂ClN₃

7/4H₂O

.

23h

C₁₃H₁₂ClN₃

1/5H₂O

62.62 5.01 16.86 63.83 5.25 16.73

23i

C₁₃H₁₂BrN₃

C₁₅H₁₈N₂O₂

C₁₆H₂₀N₂O₃

C₁₆H₁₇N₃O₂

53.81 4.17 14.48 53.70 4.06 14.36

69.74 7.02 10.85 69.55 7.20 10.66

66.65 6.99 9.72 66.45 6.75 9.61

67.83 6.05 14.83 17.56 5.83 14.66

24a

24b

24c

24f

24h

24i

C₁₅H₁₇FN₂O₂65.20 6.20 10.14 65.03 6.32 10.30.

C₁₅H₁₇ClN₂O₂61.54 5.85 9.57 61.33 5.70 9.45

C₁₅H₁₇BrN₂O₂53.42 5.08 8.31 53.35 5.15 8.17

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

62.20 6.71 31.09 61.99 6.88 31.23

58.23 6.84 27.17 58.36 6.78 27.32

149

Previously, DeGraw et al.⁴⁰reportedthat 5,10-methyl-

25a

25b

C₁₄H₁₈N₆

C₁₅H₂₀N₆O

1/2H₂O

.

enetetrahydro-8,10-dideazaaminopterin (27, Fig. 1) was

a weak inhibitor to both L1210 murine leukemia DHFR

andL1210 growth in culture. Compound 27 was only

1/100 as potent as MTX in inhibiting L1210 cell growth

in culture. Our previous report also showedthat com-

pound 13 was one tenth less active than MTX against

human leukemia Hl-60 cell growth in culture. Gangjee

et al.⁴¹synthesizedthe tetrahydro analogue of 13, 5,10-

methylenetetrahydro-5-deazafolic acid (28), which was

inactive in culturedlymphoma cell growth or TS. The

inactivity of 28 was attributedto its lack of ﬂexibility,

thus leading to its inability to bind to TS. The 7-des-

methylene-5,10-methylenetetrahydro-5-deazafolic acid

(5), on the other hand, did not display any activity

against TS or glycinamide ribotide formyltransferase,

two other key enzymes of folate-mediated one-carbon

metabolism.²³However, the triamino derivative 6 was

moderately active as an inhibitor of cultured tumor cell

growth (SCC25 human squamous cell carcinoma), but

was a goodsubstrate for folylpolyglutamate synthetase.

These reports clearly demonstrated that derivatives of

aminopterin or 2,4,6-triaminopyrimidine were more

potent than the corresponding folic acid analogues or 2,6-

diamino-4-hydroxypyrimidine derivatives, respectively.

Similar results were observedin our present studies.

25c

25d

25e

25f

25g

25h

25i

C₁₅H₁₇N₇

61.00 5.80 33.20 60.84 5.98 33.39

53.32 5.43 31.09 53.54 5.66 31.40

58.32 5.94 29.15 58.33 5.84 29.23

58.32 5.94 29.15 58.02 6.05 29.33

55.17 5.62 27.58 54.92 5.73 27.40

55.17 5.62 27.58 54.96 5.60 27.35

48.15 4.91 24.07 48.45 5.01 23.83

58.12 6.62 24.21 58.02 6.75 24.43

C₁₄H₁₇N₇O₂

C₁₄H₁₇FN₆

C₁₄H₁₇ClN₆

C₁₄H₁₇BrN₆

C₁₄H₁₇N₅O

26a

26b

.

C₁₅H₁₉N₅O₂56.41 6.63 21.93 56.66 6.62 21.75

H₂O

.

26c

26f

C₁₅H₁₆N₆O

H₂O

57.31 5.77 26.74 57.11 5.66 26.88

.

C₁₄H₁₆FN₅O 54.71 5.80 22.79 54.65 6.05 22.58

H₂O

C₁₄H₁₆ClN₅O 54.99 5.27 22.91 54.57 5.32 22.88

C₁₄H₁₆BrN₅O 48.01 4.60 20.00 47.90 4.43 20.25

26h

26i

Biological Results and Discussion

Table 3 shows the cytotoxicity of a series of 5-(N-phenyl-

pyrrolidin-3-yl)-2,4,6-triaminopyrimidines (25a–i) and2,4

- diamino - (N - phenylpyrrolidin - 3 - yl) - 6(5H) - oxopyr-

imidines (26a,b,c,f,h,i) in inhibiting human colon ade-

nocarcinoma (COLO 205), lung carcinoma (H23) and

its adriamycin resistant cell line (H23/0.3), T-cell leuke-

mia (MOLT-4), promyelocytic leukemia (Hl-60),

human T-cell acute lymphocytic leukemia (CCRF-

CEM) andhuman lung carcinoma (A549) cell growth in

culture. MTX was usedfor comparison. The results

demonstrated that in the series of 2,4,6-triaminopyr-

imidine derivatives (25a–i), the halogen substitutedsub-

stances (i.e., 25e–i) exhibitedpotent cytotoxicity;

compounds 25e and 25h are more potent than MTX

(inactive at concentrations up to 100 mM) in inhibiting

of H23/0.3 cell growth with IC₅₀values of 3.47 and3.01

mM, respectively; 25f, 25h, and 25i inhibitedMOLT-4

cell growth in culture with higher aﬃnity than other

derivatives with IC₅₀values of 3.70, 1.88 and2.48 mM,

respectively. Among halogen substitutedcompounsd,

4-bromophenyl derivative 25i displayed potent cyto-

toxicity in inhibiting cell growth of culturedhuman

leukemias (MOLT-4, Hl-60 andCCRF-CEM) and

human lung carcinoma (A549) with IC₅₀values of 2.48,

1.58, 1.80 and1.07 mM, respectively. In the same series

of derivatives, compounds with electron-withdrawing

function [CN (25c) and NO₂(25d)] on the phenyl ring

were inactive. These studies also reveal that phenyl-

(25a) or 3-methoxyphenyl- (25b) derivatives have mar-

ginal cytotoxicity in inhibiting MOLT-4 cell growth in

culture only. However, all 2,6-diamino-4(3H)-oxopyr-

imidine derivatives (26a,b,c,f,h,i) are inactive at con-

centrations up to 100 mM in all testing systems. The

results demonstrated that 2,4,6-triaminopyrimidine

derivatives are more cytotoxic than 2,4-diamino-6(5H)-

oxopyrimidine derivatives. Although some 2,4,6-triamino-

pyrimidine derivatives (i.e., 25g and 25i) are better inhi-

bitors of COLO 205, H23 andH23/0.3 cell growth in

culture than MTX, these compounds are less active than

MTX in human leukemia cell growth in culture.

In situ [5-³H]-deoxyuridine tritium release assays^42,43

were usedto evaluate the eﬀect of compounds 25f,h,i

inhibition on the folate metabolic pathways in MTX-

sensitive human ﬁbrosarcoma HT-1080 cells. Table 4

shows MTX and 25f,h,i at concentrations of 0.1–1.0 mM

inhibiting the pathway involving TS in HT-1080 cell

lines. The result revealedthat the ﬂuoro-substituted

compound, 25f, was as potent as MTX, while the

chloro- andbromo-substitutedderivatives, 25h and 25i,

were less potent than MTX on the inhibition at 0.1 mM.

Leucovorin (N-5-formyltetrahydrofolate) (LV) is widely

usedto selectively rescue the serious toxic side eﬀects of

44ꢀ46

MTX andTMQ.

In particular, LV appears to pro-

tect hemopoitic progenitors against damages by MTX

or TMQ. Therefore, we studied the eﬀects of LV on

HT-1080 cell growth inhibition by MTX and 25f,h,i at

various concentrations in the presence or absence of 10

mM LV after a 24 h exposure (Fig. 2A–D). The results

showedthat MTX was more cytotoxic than 25f,h,i on

inhibiting HT-1080 cell growth in culture (Table 5). In

the presence of LV, 3–54% of HT-1080 cells were res-

cuedby MTX+LV. A partial rescue of HT-1080 cell

growth was observedwhen the cells were treatedwith

either 25h (3–23% rescue) or 25i (1–5% rescue) simul-

taneously, at high concentration (10 mM). However, no

eﬀect of LV on the rescue of HT-1080 cells growth at

high concentration of 25h and 25i. While, up to 56% of

HT-1080 cell growth was rescuedby LV+ 25f.

In conclusion, we have synthesizeda series of non-classical

antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triamino-

pyrimidine (25a–i) and2,4-diamino-( N-phenylpyrrolidin-

3-yl)-6(5H)-oxopyrimidine (26a,b,c,f,h,i) derivatives. The

cytotoxicity of these compounds was evaluated upon

their ability to inhibit of various human tumor cell

150

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

Table 1. Yields and physical data of the synthesized compounds

(%) CompdYield

mp (

^ꢁC)

95–96

Syrup

164–165

188–189

Syrup

70–71

Syrup

106–107

Syrup

89–90

108–109

170–171

187–188

Syrup

88–89

Syrup

128–129

141–142

176–177

151–152

176–177

178–179

185–186

176–177

102–103

163–163

96–97

39–140

159–160

137–138

Syrup

168–169

134–135

Syrup

137–138

Syrup

124–125

156–157

Syrup

IR KBr (cm^{ꢀ1 a}

)

Formula^b

19a

19b

19c

19d

19e

19f

19g

19h

19i

20a

20b

20c

20d

20e

20f

20g

20h

20i

21a

21b

21f

21h

21i

22a

22b

22c

22f

22h

22i

23a

23b

23c

23d

23e

23f

23g

23h

23i

24a

24b

24c

24f

24h

24i

25a

25b

25c

25d

25e

25f

25g

25h

25i

26a

26b

26c

26f

26h

26i

62

39

19

16

25

23

52

23

54

79

49

51

55

79

69

45

66

72

27

72

65

61

34

74

75

63

80

91

60

96

31

22

56

33

81

83

48

89

80

67

82

80

48

34

7

C₁₀H₁₃NO

C₁₁H₁₅NO₂

C₁₁H₁₂N₂O

C₁₀H₁₂N₂O₃

C₁₀H₁₂FNO

C₁₀H₁₂ClNO

.

C₁₀H₁₂ClNO 5/8H₂O

C₁₀H₁₂BrNO

C₁₀H₁₁NO

C₁₁H₁₃NO₂

1755 (C¼O)

1753 (C¼O)

2213 (CN), 1756 (C¼O)

1763 (C¼O)

1754 (C¼O)

1757 (C¼O)

2239 (CN)

.

C₁₁H₁₀N₂O 1/5H₂O

C₁₀H₁₀N₂O₃

C₁₀H₁₀FNO

C₁₀H₁₀ClNO

C₁₀H₁₀BrNO

C₁₃H₁₁N₃

2234 (CN)

2239 (CN)

2230 (CN)

2236 (CN)

2239 (CN)

2230 (CN)

2215 (CN)

2233 (CN)

C₁₄H₁₃N₃O

C₁₃H₁₀FN₃

C₁₃H₁₀ClN₃

C₁₃H₁₀BrN₃

C₁₅H₁₆N₂O₂

C₁₆H₁₈N₂O₂

C₁₆H₁₅N₃O₂

C₁₅H₁₅FN₂O₂

C₁₅H₁₅ClN₂O₂

C₁₅H₁₅BrN₂O₂

2258 (CN)

2230 (CN)

2256 (CN)

.

C₁₃H₁₃N₃1/10H₂O

C₁₄H₁₅N₃O

.

C₁₄H₁₂N₄1/5H₂O

2220 (CN)

2252 (CN)

C₁₃H₁₂N₄O₂

C₁₃H₁₂FN₃

C₁₃H₁₂ClN₃

2256 (CN)

2210 (CN)

2250 (CN)

2263 (CN)

2256 (CN)

2250 (CN)

2256 (CN)

2250 (CN)

2256 (CN)

.

C₁₃H₁₂ClN₃1/5H₂O

C₁₃H₁₂BrN₃

C₁₅H₁₈N₂O₂

C₁₆H₂₀N₂O₃

C₁₆H₁₇N₃O₂

C₁₅H₁₇FN₂O₂

C₁₅H₁₇ClN₂O₂

C₁₅H₁₇BrN₂O₂

C₁₄H₁₈N₆

289–290

219–220

158–159

225–226

223–224

289–291

185–186

250–251

257–258

289–290

222–223

250–251

285–286

228–229

259–260

.

C₁₅H₂₀N₆O 1/2H₂O

2210 (CN)

C₁₅H₁₇N₇

C₁₄H₁₇N₇O₂

C₁₄H₁₇FN₆

C₁₄H₁FN₆

C₁₄H₁₇ClN₆

C₁₄H₁₇BrN₆

C₁₄H₁₇N₅O

C₁₅H₁₉O₂N₅H₂O

C₁₅H₁₆N₆OH₂O

C₁₄H₁₆FN₅OH₂O

C₁₄H₁₆ClN₅O

C₁₄H₁₆BrN₅O

12

8

56

7

43

38

56

30

15

26

15

33

^aOnly the peak for CN group was shown.

^bExcept when noted, the compounds gave elemental analyses for C, H, and N with ꢂ0.4% of the theoretical values.

growth in culture. The structure–activity relationship

studies reveal that 2,4,6-triaminopyrimidines are more

active than the 2,4-diamino-6(5H)-oxopyrimidines coun-

ter parts. The latter are inactive in all testing systems.

Among these compounds, the halogen substituted deri-

vatives of 2,4,6-triaminopyrimidine (25f,h,i) exhibit

potent cytotoxicity, especially, in inhibiting human leuke-

mia (MOLT-4, Hl-60, andCCRF-CEM), even though

they are less cytotoxic than MTX. The present studies also

demonstrate that 25f,h,i inhibit the folate metabolic

pathways in HT-1080 cell lines. The ﬂuoro-substituted

derivative (25f) is as potent as MTX. Study on the

inhibition of HT-1080 cell growth in culture demon-

strates that 25f,h,i are less cytotoxic than MTX. At high

concentration HT-1080 cell growth is partially rescued

in the presence of LV (10 mM) when the cells were

1

a

Table 2. Parameters of H NMR spectra of the synthesizedcompounds

Compd2-CH

3-CH

4-CH₂

5-CH₂

ArH

Others

2

19a

3.05 (1H, d, 10.0), 3.38

(1H, dd, 4.9, 10.0)

4.39 (1H, m)

1.88 and2.02

(each 1H, m)

3.25 and3.31

(each 1H, m)

6.49 (1H, d, 8.2), 6.56

(1H, t, 7.2), 7.14 (1H, t, 7.8)

4.94 (1H, s, OH)

19b

19c

19d

19e

19f

19g

19h

19i

3.03 (1H, d, 10.0 Hz), 3.37

(1H, dd, 4.9, 10.0)

4.38 (1H, m)

4.42 (1H, m)

4.43 (1H, m)

4.36 (1H, m)

4.38 (1H, m)

4.34 (1H, m)

4.40 (1H, m)

1.89 and2.02

(each 1H, m)

3.23 and3.28

(each 1H, m)

6.00 (1H, s), 6.10 (1H, dd, 1.3, 8.0), 6.17

(1H, dd, 1.3, 6.5), 7.03 (1H, t, 8.0)

3.69 (3H, s, CH₃), 5.01

(1H, br s, OH)

3.15 (1H, d, 10.0 Hz), 3.43

(1H, dd, 4.6, 10.8)

1.94 and2.03

(each 1H, m)

3.29 and3.39

(each 1H, m)

6.58 (2H, d, 8.7), 7.51 (2H, d, 8.7)

6.61 (2H, d, 9.0), 8.05 (2H, d, 9.0)

6.67 (2H, m), 7.02 (2H, m)

5.00 (1H, s, OH)

5.10 (1H, br s, OH)

4.95 (1H, s, OH)

4.93 (1H, s, OH)

4.96 (1H, s, OH)

4.97 (1H, s, OH)

4.91 (1H, s, OH)

3.24 (1H, d, 11.0 Hz),

3.37 (1H, m)

1.94 and2.04

(each 1H, m)

3.46 (2H, m)

3.17 (1H, d, 10.1), 3.56

(1H, dd, 5.0, 10.1)

1.85 and1.99

(each 1H, m)

3.45 (2H, m)

3.10 (1H, d, 10.0) 3.37

(1H, dd, 4.9, 10)

1.88 and2.03

(each 1H, m)

3.22 and3.29

(each 1H, m)

6.46 (2H, m), 6.99 (2H, m)

3.11 (1H, dd, 2.5, 10.1), 3.65

(1H, dd, 5.1, 10.1)

1.83 and1.98

(each 1H, m)

3.24 and3.49

(each 1H, m)

6.77 (1H, m), 6.90 (1H, m), 7.16

(1H, m), 7.27 (1H, dd, 1.3, 5.4)

3.04 (1H, d, 10.2), 3.37

(1H, dd, 4.9, 10.2)

1.89 and2.03

(each 1H, m)

3.20 and3.32

(each 1H, m)

6.49 (2H, d, 8.5), 7.16 (2H, d, 8.5)

6.45 (2H, d, 8.9), 7.25 (2H,d, 8.9)

6.61 (2H, m), 7.01 (2H, m)

3.05 (1H, dd, 2.4, 10.0), 3.37

(1H, dd, 4.9, 10.2)

1.91 and2.03

(each 1H, m)

3.20 and3.33

(each 1H, m)

20a

20b

3.66 (2H, s)

3.69 (2H, s)

2.73 (2H, t, 7.2)

2.71 (2H, t, 7.3)

3.64 (2H, t, 7.3)

3.67 (2H, t, 7.3)

6.20 (1H, d, 1.5), 6.28 (1H, dd, 1.5, 8.0), 6.39

(1H, dd, 1.5, 6.5), 7.20 (1H, dd, 6.5, 8.0)

3.80 (3H, s, Me)

20c

20d

20e

20f

20g

3.76 (2H, s)

3.82 (2H, s)

3.78 (2H, s)

3.66 (2H, s)

3.73 (2H, s)

2.79 (2H, t, 7.5)

2.83 (2H, t, 8.1)

2.65 (2H, t, 7.2)

2.73 (2H, t, 7.2)

2 .48 (2H, t, 7.4)

3.79 (2H, t, 7.5)

3.84 (2H, t, 8.1)

3.69 (2H, t, 7.2)

3.64 (2H, t, 7.2)

3.59 (2H, t, 7.4)

6.62 (2H, d, 8.7), 7.53 (2H, d, 8.7)

6.59 (2H, d, 9.2), 8.18 (2H, d, 9.2)

6.80 (2H, m), 7.06 (2H, m)

6.61 (2H, m), 7.01 (2H, m)

6.64 (1H, m), 6.98 (1H, m), 7.11 (1H, m),

7.48 (1H, m)

20h

20i

3.69 (2H, s)

3.65 (2H, s)

4.37 (2H, s)

2.75 (2H, t, 7.4)

2.73 (2H, t, 7.4)

3.27 (2H, t, 6.8)

3.66 (2H, t, 7.4)

3.69 (2H, t, 7.4)

3.61 (2H, t, 6.8)

6.59 (2H, d, 8.7), 7.25 (2H, d, 8.7)

6.52 (2H, d, 8.9), 7.36 (2H, d, 8.9)

21a

6.69 (2H, m), 6.89 (1H, m. ArH),

7.32 (2H, m)

21b

4.40 (2H, s)

3.27 (2H, t, 6.8)

3.65 (2H, t, 6.8)

6.22 (1H, s), 6.30 (1H, dd, 8.0), 6.45

(1H, dd, 8.0), 7.22 (1H, t, 8.0)

3.82 (3H, s, Me)

21f

21h

21i

4.37 (2H, s)

4.38 (2H, s)

4.37 (2H, s)

4.58 (2H, s)

3.27 (2H, t, 6.8)

3.29 (2H, t, 6.7)

3.28 (2H, t, 6.8)

3.26 (2H, t, 7.1)

3.61 (2H, t, 6.8)

3.63 (2H, t, 6.7)

3.62 (2H, t, 6.8)

3.54 (2H, t, 7.1)

6.62 (2H, m), 7.02 (2H, m)

6.60 (2H, d, 8.7), 7.25 (2H, d, 8.7).

6.54 (2H, d, 8.9), 7.38 (2H, d, 8.9)

6.72 (2H, m), 6.83 (1H, m), 7.29 (2H, m)

22a

1.38 (3H, t. 7.1, Me), 4.33

(2H, q, 7.1, CH₂

(continued on next page)

Table 2 (continued)

Compd2-CH

3-CH

4-CH₂

5-CH₂

ArH

Others

2

22b

4.56 (2H, s)

3.24 (2H, t, 7.0)

3.52 (2H, t, 7.0)

6.24 (1H, s), 6.32 (1H, m), 6.38

(1H, m), 7.18 (1H, m)

1.37 (3H, t, 7.2, Me), 3.80 (3H, s, Me),

4.31 (2H, q, 7.2, CH₂)

22c

22f

22h

22i

4.44 (2H, s)

4.34 (2H, s)

4.39 (2H, s)

3.53 (2H, t, 7.0)

3.47 (2H, t, 6.2)

3.47 (2H, t, 7.2)

3.48 (2H, t, 6.2)

3.66 (2H, t, 7.0)

3.54 (2H, t, 6.2)

3.55 (2H, t, 7.2),

3.53 (2H, t, 6.2)

6.62 (2H, d, 8.4), 7.51 (2H, d, 8.4)

6.64 (2H, m), 7.00 (2H, m)

1.34 (3H, t, 7.0, Me), 4.29 (2H, q, 7.0, Me)

1.37 (3H, t, 7.0,Me), 4.31 (2H, q, 7.0, Me)

1.35 (3H, t, 7.0, Me), 4.30 (2H, q, 7.0, CH₂)

1.35 (3H, t, 7.0, Me), 4.35 (2H, q, 7.0, CH₂)

3.75 (1H, d, 8.1, CH)

6.59 (2H, d, 8.8), 7.22 (2H, d, 8.8)

6.56 (2H, d, 8.9), 7.40 (2H, d, 8.9)

23a

23b

23c

23d

23e

23f

23g

23h

t23i

24a

24b

24c

24f

24h

24i

3.34 (1H, dd, 9.8, 6.0), 3.59

(1H, dd, 7.2, 9.8)

2.97 (1H, m)

2.99 (1H, m)

3.05 (1H, m)

2.98 (1H, m)

3.02 (1H, m)

3.03 (1H, m)

3.02 (1H, m)

2.92 (1H, m)

2.95 (1H, m)

3.01 (1H, m)

2.10 and2.40

(each 1H, m)

3.36 and3.51

(each 1H, m)

6.59 (1H, d, 8.2), 6.77, (1H, t, 7.8),

7.26, (1H,t, 7.9)

3.38 (1H, m), 3.60

(1H, dd, 7.1, 9.8)

2.10 and2.40

(each 1H, m)

3.40 and3.53

(each 1H, m)

6.13 (1H, s), 6.21 (1H, m), 6.34

(1H, m), 7.16 (1H, m)

3.79 (3H, s, Me), 3.82 (1H, d, 7.5, CH)

3.86 (1H, d, 7.2, CH)

3.38 (1H, dd, 6.9, 10.0), 3.71

(1H, dd, 7.5, 10.0)

2.16 and2.48

(each 1H, m)

3.58 and3.68

(each 1H, m)

6.54 (2H, d, 8.7), 7.48 (2H, d, 8.7)

6.69 (2H, d, 9.1), 8.03 (2H, d, 9.1)

6.71 (2H, m), 6.80 (1H, m), 7.20 (2H, m)

6.52 (2H, m), 6.97 (2H, m)

3.35–3.68 (2H, m)

2.11 and2.48

(each 1H, m)

3.35–3.68

(2H, m)

3.87 (1H, d, 8.4, CH)

3.41–3.60 (2H, m)

2.07 and2.39

(each 1H, m)

3.41–3.60

(2H, m)

3.87 (1H, d, 8.4, CH)

3.32–3.51 (1H, m), 3.57

(1H, dd, 7.2, 9.5)

2.12 and2.43

(each 1H, m)

3.32–3.51

(2H, m)

3.83 (1H, d, 7.3, CH)

3.34–3.59 (4H, m, 2-CH₂

and5-CH

2.12 and2.44

(each 1H, m),

—

6.71 (2H, m), 6.80 (2H, m)

3.83 (1H, d, 8.4, CH)

)

2

3.32–3.41 (2H, m, 2-CH and5-CH),

3.60 (1H, dd, 7.7, 9.0, 2-CH)

2.13 and2.44

(each 1H, m)

3.51

(1H, m, 5-CH)

6.50 (2H, d, 8.4), 7.20 (2H, d, 8.4)

6.45 (2H, d, 8.8), 7.33 (2H, d, 8.8)

3.83 (1H, d, 7.9, CH)

3.32 (1H, dd, 5.9, 9.8), 3.59

(1H, dd, 7.2, 9.9)

2.13 and2.43

(each 1H, m)

3.38 and3.50

(each 1H, m)

3.84 (1H, d, 8.0, CH)

3.27–3.54 (4H, m, 2-CH₂

and5-CH

2.01 and2.28

(each 1H, m)

6.53 (2H, m), 6.68

(1H, m), 7.20 (2H, m)

1.31 (3H, t, 7.0, Me), 3.58 (1H, t, 7.6, CH),

4.26 (2H, q, 7.2, CH₂)

)

2

3.33–3.56 (4H, m, 2-CH₂

and5-CH

2.00 and2.28

(each 1H, m)

6.10 (1H, m), 6.18 (1H, m), 6.29

(1H, d, 8.0), 7.14 (1H, t, 8.1)

1.33 (3H, t, 7.2, Me), 3.60 (1H, d, 8.0, CH), 3.79

(3H, s, CH₃), 4.29 (2H, q, 7.2, CH₂)

)

2

3.24–3.66 (4H, m, 2-CH₂

and5-CH

2.05 and2.38

(each 1H, m)

6.52 (2H, d, 8.6), 7.46 (2H, d, 8.6)

1.34 (3H, t, 7.2, Me), 3.65 (1H, d, 7.5, CH),

4.31 (2H, q, 7.2, CH₂)

)

2

3.30–3.55 (4H, m, 2-CH₂

and5-CH

2.04 and2.30

(each 1H, m)

6.47 (2H, m), 6.94 (2H, m)

1.33 (3H, t, 7.0, Me), 3.63 (1H, d, 7.0, CH),

4.29 (2H, q, 7.0, CH₂)

)

2

3.27–3.55 (4H, m, 2-CH₂

and5-CH

2.97 (1H, m)

2.96 (1H, m)

2.02 and2.31

(each 1H, m)

6.44 (2H, d, 8.6), 7.16 (2H, d, 8.6)

6.42 (2H, d, 8.0), 7.29 (2H, d, 8.0)

1.33 (3H, t, 7.0, Me), 3.61 (1H, d, 7.4, CH),

4.28 (2H, q, 7.0, CH₂)

)

2

3.17 (1H, dd, 7.5, 9.6),

3.50 (1H, m)

2.06 and2.35

(each 1H, m)

3.31

(1H, t, 7.8) 3.40

(1H, dt, 5.4, 8.9)

1.33 (3H, t, 7.1, Me), 3.59 (1H, d, 7.6, CH),

4.29 (2H, q, 7.1, CH₂)

25a

3.12 (1H, t, 9.5), 3.49

(1H, dd, 6.5, 9.5)

3.65 (1H, m)

2.11 (2H, m)

3.01 and3.65

(each 1H, m,)

6.67 (3H, m), 7.19 (2H, m)

5.27 (2H, s, NH₂), 5.56 (4H, s, 2ꢃNH₂)

(continued on next page)

Table 2 (continued)

Compd2-CH

3-CH

4-CH₂

5-CH₂

ArH

Others

2

25b

25c

25d

25e

25f

25g

25h

25i

3.13 and3.47 (each 1H, m)

3.48 (1H, m)

3.63 (1H, m)

3.59 (1H, m)

3.45 (1H, m)

3.64 (1H, m)

3.59 (1H, m)

2.10 (2H, m)

3.02 and3.63

(each 1H, m)

6.19 (1H, s), 6.27 (2H, m), 7.07 (1H, m)

6.69 (2H, d, 8.6), 7.55 (2H, d, 8.6)

6.59 (2H, d, 8.7), 8.05 (2H, d, 8.7)

6.87 (1H, m), 6.95 (1H, m), 7.09 (2H, m)

6.70 (2H, m), 7.03 (2H, m)

3.72 (3H, s, Me), 5.19 (2H, s, NH₂),

5.47 (4H, s, 2ꢃNH₂)

7.21 (2H, br s, NH₂), 8.99 (2H, br s, NH₂),

9.04 (2H, br s, NH₂)

3.37 (1H, m, 2⁰-CH), 3.54

(2H, m, 2⁰-CH, 5⁰-CH)

2.13 and2.35

(each 1H, m)

3.26

(1H, m, 5⁰-CH)

2.82 and3.34 (each 1H, m)

2.04 and2.17

(each 1H, m)

2.76 and3.58

(each 1H, m)

5.22 (2H, br s, NH₂), 5.51

(4H, br s, 2ꢃNH₂)

5.50 (2H, br s, NH₂), 5.86

(4H, s, 2ꢃNH₂)

5.16 (2H, br s, NH₂), 5.52

(4H, br s, 2ꢃNH₂)

5.52 (2H, br s, NH₂), 6.01

(4H, br s, 2ꢃNH₂)

5.21 (2H, s, NH₂), 5.47

(4H, s, 2ꢃNH₂)

5.19 (2H, br s, NH₂), 5.45

(4H, br s, 2ꢃNH₂)

6.03 (2H, br s, NH₂), 6.91

(2H, br s, NH₂)

3.08 and3.60 (each 1H, m)

1.97 and2.12

(each 1H, m)

2.99 and3.67

(each 1H, m)

3.08 (1H, t, 9.6), 3.46

(1H, dd, 6.0, 9.6)

2.06 and2.14

(each 1H, m)

2.99 and3.67

(each 1H, m)

3.09 and3.49 (each 1H, m)

1.95 and2.16

(each 1H, m)

2.89 and3.63

(each 1H, m)

7.03 (1H, m, 1H), 7.21 (1H, m),

7.28 (1H, m), 7.40 (1H, m)

3.14 and3.44 (each 1H, m)

2.08 and2.19

(each 1H, m)

3.05 and3.61

(each 1H, m)

6.65 (2H, d, 8.8), 7.21 (2H, d, 8.8)

3.16 (1H, t, 9.8), 3.46 (1H, t, 8.8)

3.14 and3.53 (each 1H, m)

3.59 (1H, m)

2.05 and2.19

(each 1H, m)

3.07 and3.63

(each 1H, m)

6.62 (2H, d, 8.8), 7.32 (2H, d, 8.8)

0

26a

26b

26c

26f

26h

26i

3.37 (2H, m, 3 -CH and5 -CH) 1.85 and2.59

(each 1H, m)

3.18

(1H, m, 5⁰-CH)

6.50 (2H, d, 8.6), 6.55 (1H, t, 7.3),

7.14 (2H, t, 7.3)

3.19 (1H, m), 3.51 (1H, t, 8.4)

3.17 (1H, m), 3.64 (1H, t, 9.1)

3.18 (1H, m), 3.52 (1H, t, 8.7)

3.14 (1H, m), 3.51 (1H, t, 8.6)

3.12 (1H, m), 3.52 (1H, t, 9.0)

3.35 (1H, m)

1.81 and2.58

(each 1H, m)

3.21 and3.30

(each 1H, m)

6.01 (1H, s), 6.13 (2H, m), 7.02 (1H, m)

6.56 (2H, d, 8.7), 7.48 (2H, d, 8.7)

6.51 (2H, m), 7.00 (2H, m)

3.69 (3H, s, Me), 5.82 (2H, s, NH₂), 5.94

(2H, s, NH₂), 9.76 (1H, br s, OH)

3.44 (1H, m)

1.85 and2.64

(each 1H, m)

3.26 and3.47

(each 1H, m)

5.86 (2H, br s, NH₂), 5.96

(2H, s, NH₂), 9.79 (1H, br s, OH)

3.35 (2H, m, 3⁰-CH and5 -CH) 1.89 and2.58

(each 1H, m)

3.21 (1H, m, 5⁰-CH)

5.85 (2H, s, NH₂), 5.99 (2H, s, NH₂),

9.78 (1H, br s, OH)

0

3.35 (2H, m, 3⁰-CH and5 -CH) 1.88 and2.49

(each 1H, m)

3.18 (1H, m, 5⁰-CH)

6.51(2H, d, 8.8), 7.16 (2H, d, 8.8)

6.48 (2H, d, 8.8), 7.26 (2H, d, 8.8)

5.87 (2H, s, NH₂), 5.98 (2H, s, NH₂)

0

3.36 (2H, m, 3⁰-CH and5 -CH) 1.83 and2.60

(each 1H, m)

5.84 (2H, s, NH₂), 5.96 (2H, br s, NH₂),

9.78 (1H, br s, OH)

0

^aChemical shifts in ppm (d), followedby integration values, multiplicity andcoupling constants in Hz (in parentheses), in some cases, the location of the proton is indicated . When the signals appearedsuperimposed

with other signals a range is indicated, followed by the total number of H and their location (in parentheses).

154

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

Table 3. Inhibition of human tumor cell growth by 5-(N-phenylpyrrololidin-3-yl)pyrimidine derivatives

1

CompdR

R²

Inhibitory concentration (IC₅₀, mM)

LV

rescue

COLO 205

H23

H23/0.3

MOLT-4

Hl-60

CCRF-CEM

A549

25a

25b

25c

25d

25e

25f

25g

25h

25i

26a

26b

26c

26f

26h

26i

H

3-OMe

4-CN

4-NO₂

2-F

NH₂

OH

>100

79.4

>100

3.47

21.5

11.8

>100

18.9

ND

8.59

ND

4.62

1.58

ND

0.058

ND

18.6

ND

12.3

1.80

ND

0.022

ND

7.28

ND

5.93

1.07

ND

0.083

ND

++++

ND

++

ND

90.0

>100

4-F

>100

80.3

3.70

12.3

1.88

2.48

2-Cl

4-Cl

4-Br

H

4-CN

4-NO₂

4-F

49.5

44.1

55.5

>100

23.3

3.01

53.9

>100

72.6

OH

>100

ND

4-Cl

4-Br

OH

>100

0.0817

ND

+++++

MTX

Table 4. Inhibition of in situ folate metabolic pathway (tritium

release) by compound 25f, 25h, and 25i in human ﬁbrosarcoma HT-

1080 cells

Table 5. The eﬀects of leucovorin on human ﬁbrosarcoma HT-1080

cell growth inhibition by MTX andcompounds 25f, 25h and 25i

CompdLV

IC

₅₀(mM)

% Rescue in survival in the

presence of LV (10 mM) at

0.00001–10 mM of antifolate

Cell

Tritium release (% of control)

25f 25h

MTX

25i

MTX

25f

ꢀ

+

ꢀ

1.14

0.1

1.0

56

0.1

1.0

63

0.1

1.0

70

0.1

1.0

44

> > >10

2.45

> > >10

8.80

>10.0

2.45

> > >10

3–54

2–56

3–23

1–15

(mM)

HT-1080

78

80

65

+

ꢀ

25h

25i

+

ꢀ

+

treatedwith 25h or 25i simultaneously. However, the

HT-1080 cell growth was rescuedwhen cells were trea-

tedwith 25f+LV. The results indirectly prove that

25f,h,i inhibit the DHFR activity in HT-1080 cell lines.

These studies demonstrate that the target compounds

are diﬀerent from classical folate antagonists and may

47,48

be involvedin the reducedfolate transport system.

Further antitumor properties of these compounds need

to be investigated.

Experimental

Column chromatography was performedwith silica gel

G60 (70–230 mesh, ASTM; Merck) as the stationary

phase, while thin-layer chromatography was carriedout

on silica gel G60 F254 (Merck) using short-wavelength

UV light for visualization. Melting points were deter-

minedon a Fargo melting point apparatus andwere

uncorrected. Elemental analyses were performed on a

Heraeus CHN-O Rapidinstrument andthe results (for

C, H, andN), indicatedin Table 1, were within ꢂ0.4%

of the calculatedvalues. The physical properties andthe

yieldof each compoundwere also listedin Table 1. The

¹H NMR spectra of all compounds were recorded on a

Brucker AMX-400 spectrometer (400 MHz) with Me₄Si

as the internal standard. The COSY experiments were

recorded using standard pulse sequences in phase-sensi-

tive mode. Chemical shifts were reported in ppm (d),

Figure 2. Inhibition eﬀects of MTX, compounds 25f, 25h, and 25i and

in the presence of LV (10 mM) against human ﬁbrosarcoma HT-11 cell

growth in culture after 24 h exposure.

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

155

and the signals were described as s (singlet), d (doublet),

dd (doublet of doublets), t (triplet), q (quartet), m

(multiplet) andbr (broad). Values reportedfor coupling

constant are ﬁrst-order. IR spectra were measured on

Infraredspectrophotometer, Model 500, Buck Scientiﬁc

presence of KF (for compounds 21e,g). Products

21a,b,f,h,I, precipitatedduring the course of the reaction

were isolated in a stable solid form. The crude products

21c,d, which did not precipitate out and contained a

small amount of starting material along with by-pro-

ducts, were used directly for the next reaction. 3-

[Cyano(ethoxycarbonyl)methylene]-1-phenylpyrrolidine

derivatives (22a,b,c,f,h,i) were synthesizedby reacting

20a–i with ethyl cyanoacetate in dry ether in the pre-

sence of N,N-dimethylethanolamine. The procedure for

the preparation of 3-(dicyanomethylene)-1-phenyl-

pyrrolidine (21a) is representative. Ten drops of DBU

were added slowly to a mixture of 20a (7.08 g, 44.0

mmol) andmalononitrile (8.81 g, 88.0 mmol) in dry

ether (20 mL) with vigorous stirring at 0 ^ꢁC. After 10

min, the reaction mixture was stirredat room tempera-

ture for an additional 8 h andthe resulting solidproduct

21a was collectedby ﬁltration andrecrystallizedfrom

MeOH/CHCl₃(3.28 g; 72%) as light green crystals.

Following the same procedure as that for the synthesis of

21a, the compounds 21b–i and 22a,b,c,f,h,i were prepared.

1

Inc. The IR and H NMR spectra of each compound

are listedin Tables 1 and2, respectively. The repre-

sentative methodfor the synthesis of each series of

compounds are described as follows:

Derivatives of 1-phenyl-3-pyrrolidinol (19a–i). The gen-

eral procedure used was similar to that reported pre-

viously.³⁷The procedure for the preparation of 1-

phenyl-3-pyrrolidinol (19a) is representative. 1,4-

Dibromo-2-butanol (17, 35.0 g, 0.16 mol) was added

dropwise to a blend of aniline (18a, 14.5 g, 0.16 mol),

K₂CO₃(65.0 g, 0.48 mol) andtriethyl phosphate (150

mL) under nitrogen at room temperature and then a

reaction mixture was heatedat 150 ^ꢁC (oil bath tem-

perature) for 6 h. After cooling, the mixture was ﬁltered

andthe solidcake was washedwith ethyl acetate (200

mL). The combinedﬁltrate andwashings were evapo-

rated in vacuo and the residue was diluted with water

(600 mL) andextractedseveral times with ethyl acetate.

The combinedorganic extracts were washedwith water,

dried over Na₂SO₄andevaporatedin vacuo to dryness.

The dark residue was chromatographed on a silica gel

column (5ꢃ30 cm) using hexane/EtOAc (20:1 v/v) as the

eluent. The major UV-absorbing fraction was con-

centratedto aﬀord 19a, 15.7 g (62%). Compounds 19b–i

were preparedusing the same procedure as 19a.

Derivatives of 3-(dicyanomethyl)-1-phenylpyrrolidine (23a–

i) and 3-[cyano(ethoxycarbonyl)methyl]-1-phenylpyrrolidine

(24a,b,c,g,h,i). All compounds of these two groups were

preparedby the reduction either 21a–i or 22a,b,c,g,h,i

with sodium cyanoborohydride in acetic acid. Products,

23b,e,g and 24a,b,c,f,h,i, were isolatedas syrup by liquid

column chromatography (SiO₂, hexane/EtOAc, 8:1 v/v).

Compounds 23a,c,d,f,h,i were isolatedas crystals by

direct crystallization after ﬁltration and removal of sol-

vent from the reaction mixture. The procedure for the

preparation of 3-(dicyanomethyl)-1-phenylpyrrolidine

(23a) is representative. Sodium cyanoborohydride (0.66

g, 10.5 mmol) was added portionwise to a suspension of

21a (2.20 g, 10.5 mmol) in acetic acid(5.0 mL) at room

temperature. After stirring for 30 min, the clear solution

was evaporatedin vacuo to dryness andco-evaporated

several times with toluene. The residue was dissolved in

EtOAc (100 mL), washedwith water (20 mL ꢃ2), dried

over Na₂SO₄, andevaporatedin vacuo to dryness. The

residue was crystallized from EtOH to give 23a: (1.32 g;

60%). By following the same procedure as that for the

synthesis of 23a, compounds 23b–i and 24a,b,c,g,h,i

were prepared. The yields of compounds 23d,e,g were

calculatedbasedon 20d,e,g, respectively.

Derivatives of 1-phenyl-3-pyrrolidinone (20a–i). All com-

pounds in this group were prepared by following the

procedure described previously.³⁷The procedure for the

preparation of 1-phenyl-3-pyrrolidinone (20a) is repre-

sentative. Triﬂuoroacetic acid(3.7 mL, 48 mmol) was

added dropwise to a mixture of 19a (15.6 g, 95 mmol) in

dry benzene (600 mL) containing N,N-dicyclohexyl-

carbodimide (DCC, 39.3 g, 190 mmol), anhydrous

DMSO (7.0 mL) and anhydrous pyridine (8 mL) with

vigorous stirring under nitrogen at 0 C for 30 min. The

mixture was stirredat room temperature for 24 h. After

evaporation of benzene under reduced pressure a solid

was removedby ﬁltration andwashedthoroughly with

ethyl acetate (300 mL). The combinedﬁltrate and

washings were washedwith H ₂O followedwith 5%

copper(II)-sulfate aqueous solution to remove pyridine.

It was then dried over Na₂SO₄, ﬁlteredandevaporated

in vacuo to dryness. The residue was crystallized from

MeOH/CHCl₃to aﬀordwhite needle crystals 20a: 17.1

g (79%). Compounds 20a–i were preparedusing the

same procedure as for the synthesis of 20a.

Derivatives of 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triamino-

pyrimidine (25a–i) and 2,4-diamino-5-(N-phenylpyrrol-

idin-3-yl)-6(5H)-oxopyrimidine

(26a,b,c,g,h,i).

All

compounds of these two groups were prepared by fol-

lowing the procedure as described previously.^27,37The

procedure for the preparation of 5-(N-phenylpyrrolidin-

3-yl)-2,4,6-triaminopyrimidine (25a) is representative. A

mixture of 23a (1.20 g, 5.68 mmol), guanidine carbonate

(1.10 g, 5.68 mmol) in diglyme (10 mL) was heated at

140 ^ꢁC (oil bath temperature) with vigorous stirring for

1 h. After cooling, the reaction mixture was triturated

with hexane (5ꢃ10 mL) to remove diglyme. The gummy

residue was crystallized from MeOH to give 25a, 0.73 g

(48%). Using the same procedure as for the synthesis

of 25a, the compounds 25b–i and 26a,b,c,g,h,i were

prepared.

Derivatives of 3-(dicyanomethylene)-1-phenylpyrrolidine

(21a–i) and 3-[cyano(ethoxycarbonyl)methylene]-1-phenyl-

pyrrolidine (22a,b,c,f,h,i). All compounds in these two

groups were prepared by following the procedure descri-

bedpreviously, ^23,37with some modiﬁcations. Derivatives

of 3-(dicyanomethylene)-1-phenylpyrrolidine (21a–i)

were preparedby the reaction of 1-phenylpyrrolidinones

with malononitrile in dry ether in the presence of DBU

(for compounds 21a,b,c,f,h,i) or in dry EtOH in the

156

Y.-L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 145–157

Biological assays

References and Notes

Cytotoxicity assays. XTT-tetrazolium assay determined

the eﬀects of the compounds on cell growth were deter-

minedin all human tumor cells, namely human colon

adenocarcinoma (COLO 205), lung carcinoma (H23)

andits adriamycin resistant cell line (H23/0.3), T-cell

leukemia (MOLT-4), promyelocytic leukemia (Hl-60),

human T-cell acute lymphocytic leukemia (CCRF-CEM),

andhuman lung carcinoma (A549) in a 72 h incubation,

as described by Scundieo et al.⁴⁹After treatment with

phenazine methosulfate-XTT solution at 37 ^ꢁC for 6 h,

absorbance at 450 and630 nm was detectedwith a

microplate reader (EL 340, Bio-Tek Instruments Inc.,

Winooski, VT, USA). Six to seven concentrations of

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each compoundwere used. The IC

relationships of the compounds for antitumor activity

anddose–eﬀect

50

50,51

were calculatedby a meidan-eﬀect plot,

using a

computer program on an IBM-PC workstation.⁵¹

Tritium release assay. The whole cell in situ folate

metabolic pathway inhibition assay,⁴²as modiﬁed by

Rodenhuis et al.⁴³was usedto measure the eﬀect of

25f,h,i with or without leucovorin (LV) in HT-1080

cells. Cells were exposedto various concentrations of

25f,h,i in the presence or absence of 10 mM LV either for

3 h or for 4 h incubation in drug-free medium. 2⁰-[5-³H]-

Deoxyuridine was then added at a ﬁnal concentration of

2 mCi/mL and its conversion to thymidylate was deter-

minedby ³H release at 0, 15, 30, and45 min. At these

times, a 100 mL aliquot of cells was placedin 200 mL of

4% (w/w) trichloroacetic acidwith 15% charcoal to

stop the reaction. The mixture was centrifugedfor 5

min, andthe supernatant (100 mL) was added to 5 mL

of Ecolume (ICN) scintillation ﬂuidandcountedin a

scintillation counter (Beckman Model 5801). A blank

consisting of medium without cells and drugs was used

for backgroundsubtraction. The results of the scintilla-

tion counts were analyzedby calculating the slope of the

³H release using linear regression. Drug inhibition was

expressedas a percentage of the slope of untreated

control cells in assay.

14. Bigham, E. C.; Hodson, S. J.; Mallory, W. R.; Wilson, D.;

Duch, D. S.; Smith, G. K.; Ferone, R. J. Med. Chem. 1992, 35,

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18. Rosowsky, A.; Bader, H.; Wright, J. E.; Moran, R. G. J.

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Leucovorin rescue studies.⁴⁶Exponentially growing a

MTX-sensitive human ﬁbrosarcoma cell line (HT-

1080) (5ꢃ10⁵cells/mL) were exposedto various

concentrations of MTX and 25f,h,i for 24 h in the

presence or absence of 10 mM LV. After the medium

was removed, the cells were washed twice with cold

phosphate buﬀeredsaline (PBS), andfresh drug-free

medium was added. Growth was followed for 94 h

andcells were countedusing a Moedl ZB Coulter

19. Grivsky, E. M.; Lee, S.; Sigel, C. W.; Duch, D. S.; Nichol,

C. A. J. Med. Chem. 1980, 23, 327.

20. Taylor, I. W.; Slowiaczek, P.; Friedlander, M. L. Cancer

Res. 1985, 45, 978.

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41, 4533.

22. Lin, J. T.; Bertino, J. R. J. Clin. Oncol. 1987, 5, 2032.

23. Rosowsky, A.; Papoulis, A. T.; Forsch, R. A.; Queener,

S. F. J. Med. Chem. 1999, 42, 1007.

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25. Manchand, P. S.; Rosen, P.; Belica, P. S.; Olivea, G. V.;

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26. Russell, P. B.; Hitchings, G. H. J. Am. Chem. Soc. 1951,

73, 3763.

Counter. The percentage of growth inhibition andIC

values were determined from the growth inhibition

data.⁵²

50

27. de Jager, R.; Dupont, D.; Rodzynek, J. J.; Dorelet, C.;

Largrange, G. Cancer Chemother. Pharmacol. 1981, 6, 75.

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Acknowledgements

The research is supportedby part of the Institute of

Biomedical Sciences, Academia Sinica, Taipei, Taiwan

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Non-classical antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines and 2,4-Diamino-6(5H)-oxopyrimidines, synthesis and antitumor studies

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