Antitumor studies. Part 3: Design, synthesis, antitumor activity, and molecular docking study of novel 2-methylthio-, 2-amino-, and 2-(N-substituted amino)-10-alkyl-2-deoxo-5-deazaflavins

Bioorganic & Medicinal Chemistry 15 (2007) 6336–6352

Antitumor studies. Part 3: Design, synthesis, antitumor activity,

and molecular docking study of novel 2-methylthio-, 2-amino-,

and 2-(N-substituted amino)-10-alkyl-2-deoxo-5-deazaﬂavins

Hamed I. Ali,^aNoriyuki Ashida^band Tomohisa Nagamatsu^a,*

^aDepartment of Drug Discovery and Development, Division of Pharmaceutical Sciences, Graduate School of Medicine,

Dentistry and Pharmaceutical Sciences, Okayama University 1-1-1, Tsushima-Naka, Okayama 700-8530, Japan

^bBiology Laboratory, Research and Development Division, Yamasa Shoyu Co., Choshi, Chiba 288-0056, Japan

Received 11 June 2007; revised 28 June 2007; accepted 29 June 2007

Available online 4 July 2007

Abstract—Various novel 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins have been synthesized by reaction of 6-(N-alkylanilino)-2-

methylthiopyrimidin-4(3H)-ones with Vilsmeier reagent. The similar 2-(N-substituted amino) derivatives were prepared by nucleo-

philic replacement reaction of the 2-methylthio moiety by appropriate amines. The 2-oxo derivatives (i.e., 5-deazaﬂavins) were

obtained by acidic hydrolysis of the 2-methylthio derivatives. The antitumor activities against CCRF-HSB-2 and KB cells and

the antiviral activities against HSV-1 and HSV-2 have been investigated in vitro, and many compounds showed promising antitu-

mor activities. Furthermore, AutoDock molecular docking into PTK has been done for lead optimization of these compounds as

potential PTK inhibitors. Whereas, the designed 2-deoxo-5-deazaﬂavins connected with amino acids at the 2-position exhibited the

good binding aﬃnities into PTK with more hydrogen bonds.

1. Introduction

tral to all organisms and the tyrosine kinase has been

found to participate in human neoplastic diseases. Tyro-

sine kinase inhibitors and their potentials in the clinical

applications are well documented by dramatic examples

such as Gleevec, Iressa, and Herceptin. Several tyrosine

kinase inhibitors are undergoing human trials and sev-

eral are in the pipeline of drug discovery.¹⁰Molecular

docking has been a focus of attention for many years.

Generally speaking, today’s ﬂexible docking programs

such as AutoDock are able to predict protein-ligand

complex structures with reasonable accuracy and

speed.¹¹One of the most reliable, robust, and popular

energy-based docking packages is AutoGrid/AutoDock

because it allows a very eﬃcient docking of ﬂexible li-

gands (e.g., substrates, drug candidates, inhibitors, pep-

tides, etc.) onto receptors (e.g., enzymes, antibodies,

nucleic acids, etc.).¹²

Design and synthesis of 5-deazaﬂavin and ﬂavin analogs

as potential cancer chemotherapeutic agents have been

in progress in our laboratory.^1–6Recently, we have re-

ported the signiﬁcant potential antitumor activities of

10-substituted 2-deoxo-2-phenyl-5-deazaﬂavins and ﬂa-

vin-5-oxides against NCI-H 460, HCT 116, A 431,

CCRF-HSB-2, and KB cells (Scheme 1).¹5-Amino-5-

deazaﬂavins also revealed antiproliferative activity

against L1210 and KB cells.⁷10-Aryl-5-deazaﬂavin

derivatives were preliminarily reported to act as inhibi-

tors against E3 activity of HMD2 in tumors that retain

wild-type P53.⁸Actually, 5-deazaﬂavins have attracted

great interest because of their homologues as potential

riboﬂavin antagonists, bio-reductive cofactors for the

naturally occurring ﬂavins, for example, F₄₂₀, and po-

tent antitumor agents.⁹The role of tyrosine kinase in

the control of cellular growth and diﬀerentiation is cen-

Despite the large number of publications on chemistry

of 5-deazaﬂavin and related compounds, the informa-

tion of 2-methylthio- and 2-(N-substituted amino)-2-

deoxo-5-deazaﬂavins is none yet. The above circum-

stances led us to seek a convenient synthetic route to

the 2-methylthio- and 2-(N-substituted amino)-2-deo-

xo-5-deazaﬂavins to search their potential antitumor

Keywords: Antitumor activity; 5-Deazaﬂavin; AutoDock; Protein tyro-

sine kinase.

*

Corresponding author. Tel.: +81 86 251 7931; fax: +81 86 251

7926; e-mail: nagamatsu@pheasant.pharm.okayama-u.ac.jp

doi:10.1016/j.bmc.2007.06.058

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6337

obtained 4,6-dichloro-2-methylthiopyrimidine.¹⁷The

key intermediate, 6-(N-monoalkylanilino)-2-methylthio-

pyrimidin-4(3H)-ones (1a–j), was synthesized by the

treatment of 6-chloro-2-methylthiopyrimidin-4(3H)-

one^14,16with appropriate N-alkylanilines in n-butanol

under reﬂux for 12–72 h in 55–83% yields as shown in

Scheme 2. The commercially unavailable N-monome-

thylated anilines were prepared from the suitable aniline

derivatives in two steps. Thus, the N-formylation of ani-

lines with ethyl formate under reﬂux for 15 h gave the

corresponding N-arylformamides, followed by reduction

of the N-arylformamides using LiAlH₄in dry THF to

aﬀord the corresponding N-monomethylated anilines.¹⁸

The intended 10-alkyl-2-deoxo-2-methylthio-5-deazaf-

lavins (2a–j) were prepared by the reaction of 1a–j with

Vilsmeier reagent (N,N-dimethylformamide–phosphoryl

chloride) at 90 °C for 0.5–2 h to aﬀord the products as

yellow needles in 71–99% yields. Since the direct cycliza-

tion of 6-(N-methylanilino)-4-oxo-2-thioxo-1,2,3,4-tet-

rahydropyrimidines was not successful, the 2-thioxo

moiety should be protected by methylation before the

cyclization with Vilsmeier reagent.

Scheme 1. 5-Deazaﬂavins, 2-deoxo-5-deazaﬂavins, and 2-deoxo-ﬂavin-

5-oxides.

activities. In this study, we describe the facile synthesis

of 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins as key

compounds for antitumor active derivatives. These com-

pounds were used as versatile intermediates for synthesis

of the various amino coupling derivatives by nucleo-

philic substitution between 10-alkyl-2-deoxo-2-methyl-

thio-5-deazaﬂavins and suitable primary and

secondary alkylamines. Furthermore, they were used

in a novel route for the preparation of 5-deazaﬂavins

{pyrimido[4,5-b]quinoline-2,4(3H,10H)-diones} in good

yields. These compounds were investigated for their

in vitro antitumor and antiviral activities. In fact, many

of the synthesized compounds showed promising antitu-

mor activities against CCRF-HSB-2 and KB cells. Also

the SAR study was carried out with the aid of molecular

modeling using AutoDock for the aim to get designed

compounds possessing higher binding aﬃnities into

c-kit receptor of PTK. The correlation between the

growth inhibitory activities (IC₅₀, lg/mL) of the synthe-

sized 5-deazaﬂavins against tumor cells and the Auto-

Dock binding free energies was investigated, where a

fairly good correlation for some compounds was ob-

tained. Also the computationally designed 10-alkyl-2-

deoxo-5-deazaﬂavins connected with amino acids at

the 2-position exhibited preferential binding aﬃnities

into PTK with more hydrogen bonds.

The preparation for such N-substituted amino deriva-

tives by reaction of alkylthio derivatives with appropri-

ate primary or secondary amines has long been utilized

in heterocyclic chemistry. Many of these reactions re-

quire rather strenuous conditions and are usually carried

out in a stainless steel sealed vessel except when amines

of high boiling point are used.¹⁹The pyrimidine moieties

of 5-deazaﬂavins to undergo nucleophilic substitution

reactions on the carbons adjacent to ring nitrogens are

well authenticated and readily explicable in view of the

p electron-deﬁcient nature. Therefore, the various amin-

ations were applied for the replacement of 2-methylthio

group by appropriate amines for the purpose of synthe-

ses of 2-amino- and 2-(substituted amino)-10-alkyl-2-

deoxo-5-deazaﬂavins {2-amino- and 2-(substituted ami-

no)-10-alkylpyrimido[4,5-b]quinolin-4(10H)-ones} (3a–

e, 4a–c, 6, and 7a–q) as shown in Scheme 3. Namely,

10-alkyl-2-deoxo-2-dimethylamino-5-deazaﬂavins (3a–

e) were prepared by heating the suitable 10-alkyl-2-deo-

xo-2-methylthio-5-deazaﬂavins (2) with aqueous dim-

ethylamine in a stainless steel sealed vessel at 135 °C

for 4 h to aﬀord the corresponding products as yellow

needles in 59–96% yields. Whereas, the 10-alkyl-2-ami-

no-2-deoxo-5-deazaﬂavins (4a–c) were prepared by fu-

sion of the suitable 2 with ammonium acetate at 160–

165 °C for 0.5–3 h to aﬀord the corresponding products

as yellow needles in 57–77% yields. A similar treatment

of 2-deoxo-10-methyl-2-methylthio-5-deazaﬂavin (2a)

with n-butylammonium acetate (prepared in situ) at

150–165 °C for 1–3 h resulted in the formation of 2-n-

butylamino-2-deoxo-10-methyl-5-deazaﬂavin (6) as a

yellow crystalline solid in 91% yield. Moreover, a variety

of 2-(substituted amino)-10-alkyl-2-deoxo-5-deazaﬂav-

ins (7a–q) were prepared by the facile method involving

reﬂux of compound 2 with an appropriate morpholine,

piperidine, N-methylpiperazine, N-methyl-N-ethanol-

amine, ethanolamine, or n-octylamine in n-butanol for

2–5 h. All products 7a–q were obtained as bright yellow

needles in quite good yields of 70–99%. For all the

above-mentioned reactions which involve treatment

2. Results and discussion

2.1. Chemistry

The requisite 6-chloro-2-methylthiopyrimidin-4(3H)-

one was prepared by treatment of 4-amino-6-chloro-2-

methylthiopyrimidine¹³with NaNO₂in glacial acetic

acid according to the method of Israel et al.¹⁴The 4-

amino-6-chloro-2-methylthiopyrimidine was obtained

by reaction of thiourea and ethyl cyanoacetate, followed

by methylation of the produced 4-amino-6-hydroxy-2-

mercaptopyrimidine with dimethylsulfate and by chlori-

nation with phosphoryl chloride. 6-Chloro-2-methyl-

thiopyrimidin-4(3H)-one was also prepared by

alternative route from S-methylation of thiobarbituric

acid with MeI,¹⁵followed by chlorination with phos-

phoryl chloride,¹⁶and partially basic hydrolysis of the

6338

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

Scheme 2. General methods for the preparation of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones (1a–j) and 10-alkyl-2-deoxo-2-methylthio-

5-deazaﬂavins (2a–j). Reagents and conditions: (a) n-Butanol, reﬂux 12–72 h; commercially unavailable N-monoalkylanilines were prepared by

reﬂuxing with HCOOEt for 15 h, followed by reduction with LiAlH₄in THF at 0 °C for 5–12 h; (b) Vilsmeier reagent (DMF-POCl₃), 90 °C, 1–3 h.

with diﬀerent amines, the excess volatile amines were re-

moved by concentration in vacuo. Whereas, the amines

of high boiling point were removed by leaching from the

residue with water or ethanol for piperidine, morphol-

ine, N-(2-hydroxyethyl)-N-methylamine, and N-methyl-

piperazine, and petroleum ether for n-octylamine to

get the amine-free products. Acidic hydrolysis of some

10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2) in 5 N

hydrochloric acid by heating under reﬂux for 1–2 h gave

the corresponding 10-alkyl-5-deazaﬂavins (5a–c) as yel-

low needles in 78–91% yields as shown in Scheme 3.

nal of the 2-methylthio group, which were assigned for

compounds 2a–j at 2.56–2.63 ppm. The 2-(substituted

amino) derivatives (3, 4, 6, and 7) and 5-deazaﬂavins

(5) showed a characteristic singlet signal of the C₅-proton

in the lower ﬁeld at 8.77–9.12 and 8.93–9.0 ppm, respec-

tively. The methine protons (8.77–9.12 ppm) at 5-posi-

tion for 3, 4, 6, and 7 appeared in upper ﬁeld than

those (9.09–9.63 ppm) of 2a–j, due to the higher electron

donating properties of the 2-(substituted amine) groups

in comparison with that of the 2-methylthio group.

1

Regarding the H NMR of 10-alkyl-2-deoxo-2-dimeth-

UV–vis, IR, and NMR spectra and elemental analyses

were used for determination and identiﬁcation of the

newly assigned structures. The structures of the key

intermediates 1a–j were conﬁrmed in particular by the

presence of a proton resonance at the 5-position as a sin-

ylamino-5-deazaﬂavins (3a–e), it was noticed that the

N₁₀-methyl derivatives (3a,c–e) exhibited the equivalent

N₂-dimethyl groups as an equivalent singlet signal of 6H

integral almost at 3.33 ppm. Whereas, the N₁₀-ethyl

derivative (3b) exhibited the nonequivalent N₂-dimethyl

groups as two singlet signals at 3.34 and 3.48 ppm due to

the steric hindrance of longer chain of ethyl group at the

N-10 position.

1

glet signal at 4.62–5.08 ppm in H NMR spectra. The

cyclized 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins

(2a–j) showed a characteristic singlet signal due to the

C₅-proton in the lower ﬁeld at 9.09–9.63 ppm. It is

implying that the methine at the 5-position of 2a–j is

more electron-deﬁcient than the other methines. Any

substituent at the benzene ring led this singlet signal of

the C₅-proton to the slight upﬁeld, except for 2j (2-deo-

xo-6,9-dimethoxy-10-methyl-2-methylthio-5-deazaﬂavin),

which exhibits this signal in more downﬁeld at

9.63 ppm. This can be explained by the possible forma-

tion of a hydrogen bond between the oxygen atom of

6-OMe moiety and the proton at the 5-position. This

suggestion is supported by the similar eﬀect between

9-OMe group and the N₁₀-methyl moiety of 2j, that is,

the chemical shift of methyl group at the 10-position

shown in more downﬁeld region at 4.48 ppm in compar-

ison with N₁₀-methyl moieties of other derivatives ap-

peared in the range of 4.21–4.35 ppm. This eﬀect was

also clearly observed in the UV spectrum of 2j, which

indicated some hypsochromic shift, compared with other

Interestingly, the phenomenon of reversible interconver-

sion of two isomers at 27 °C in case of the 2-mono-

alkylamino (secondary amino) derivatives (6, and 7p,q)

1

was observed as tautomerism in H NMR spectra. The

twin overlapped spectra of approximately 1:2 or 2:1 ra-

tio of the 2-monoalkylamino and 2-monoalkylimino

tautomers in (CD₃)₂SO were obtained for the two reso-

nance species (tautomers). Especially, such duplicated

spectra were observed in the range of 4.0–9.0 ppm based

on the tautomers of the guanidine adjacent to carbonyl

group. At higher temperature of 90–100 °C, the coales-

cence of the duplicated spectrum was observed to pro-

duce the single spectrum. As indicated in Figure 1(A)

for compound 6, the ¹H NMR spectrum at 27 °C exhib-

ited two singlet signals of C₅-H and N₁₀-Me at 8.81 and

8.87 ppm and at 4.05 and 4.14 ppm, respectively, and

other protons at 6, 7, 8, and 9-positions were also dupli-

cated. On the other hand, in the case of 100 °C, the sin-

glet signal at 8.80 and 4.07 ppm, which was attributable

to the C₅-H and N₁₀-Me, respectively, appeared as

1

spectra of 2. Considering the H NMR spectra, the 2-

deoxo-5-deazaﬂavin derivatives (3–7) are characterized

by disappearance of the strong characteristic singlet sig-

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6339

Scheme 3. General methods for the preparation of 10-alkyl-2-deoxo-2-(substituted amino)-5-deazaﬂavins (3a–e, 6, and 7a–q), 10-alkyl-2-amino-2-

deoxo-5-deazaﬂavins (4a–c), and 10-alkyl-5-deazaﬂavins (5a–c). Reagents and conditions: (a) 50% aqueous NHMe₂, steel sealed tube, 135 °C, 4 h;

(b) CH₃COONH₄, 160–165 °C, 0.5–3 h; (c) 5 N HCl, reﬂux, 3–5 h; (d) n-Butylammonium acetate, fusion, 150–165 °C, 1–3 h; (e) appropriate amines,

n-butanol, reﬂux, 2–5 h; (f) computationally designed structures.

shown Figure 1(B) and the duplicated spectral signals

for other protons also coalesced. The 2-n-butylimino

tautomer is proposed predominantly at higher tempera-

ture because it has a heat of formation (PM3-Mozyme)

of 36.23 kcal/mol which is higher than that of 2-butyla-

mino tautomer (16.95 kcal/mol). This phenomenon is

mainly attributed to the presence of a secondary amine

at the 2-position, whereas it does not take place in case

of 2-substituted primary and teritiary amines, for exam-

ple, compounds 4a–c and 7a–o, respectively.

gether with two absorption shoulders at 259–275 and

287–293 nm. Whereas, the tricyclic 5-deazaﬂavins (2a–

j) showed ﬁve absorption maxima at 213–231, 259–

275, 278–291, 311–351, and 403–450 nm. The absorp-

tion band at 259–275 nm was shown as a maximum

absorption peak or a shoulder. Compound 2j indicated

the characteristic blue shift (hypsochromic shift) in all

wavelength regions in comparison with those of 2a–i.

This may be attributed to the hydrogen bond formation

between the oxygen atom of 6-OMe moiety and C₅-pro-

ton of 2j. This hinders the delocalization of the lone pair

on oxygen of 6-OMe to the ring system by the hydrogen

bond. The UV spectra of 2-(substituted amino)-10-al-

kyl-2-deoxo-5-deazaﬂavins (3, 4, 6, and 7) showed ﬁve

absorption maxima at 220–225, 268–288, 318–326,

407–428, and 424–454 nm, together with an absorption

shoulder at 383–404 nm, except for compound 7n. All

5-deazaﬂavin analogs (2–7) showed yellow color owing

to the presence of absorption maximum at 400–440 nm

in the longest wavelength. Generally, the UV spectra

The structural identity of the prepared compounds was

veriﬁed by UV–vis absorption spectra. The spectra for

10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2a–j) lo-

cated in shorter wavelength region in comparison with

those for previously reported 2-deoxo-2-phenyl-5-deaza-

ﬂavin derivatives,¹which have more conjugated ring sys-

tem. The UV absorption spectra of the 6-(N-

alkylanilino)-2-methylthiopyrimidin-4(3H)-ones (1a–j)

showed one absorption maximum at 239–246 nm to-

6340

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

Figure 1. The ¹H NMR spectra of compound 6 at 27 °C (A) and at 100 °C (B) in (CD₃)₂SO.

of 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2)

exhibited longer wavelength than those of 2-(substituted

amino)-10-alkyl-2-deoxo-5-deazaﬂavins and 5-deazaf-

lavins (3–7) as represented in Figure 2 for compounds

1a, 2a, 5a, and 7a. This is attributed to the S-atom which

causes a generally red shift (bathochromic shift) in the

spectrum due to its easier polarizability.²⁰Thus, the

spectrum of 2-deoxo-10-methyl-2-methylthio-5-deaza-

ﬂavin (2a) exhibited the bathochromic shift of ca.

20 nm longer wavelength than that of 5-deazaﬂavin

(5a) in all UV and visible regions.

man T-cell acute lymphoblastoid leukemia cell line

(CCRF-HSB-2) and human oral epidermoid carcinoma

cell line (KB). The antitumor agent, cytosine arabino-

side (Ara-C), was used as positive control.

As can be seen in Table 1, although all compounds of

10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2) and

10-alkyl-2-deoxo-2-dimethylamino-5-deazaﬂavins

(3)

inferior antitumor activities compared to Ara-C, they

have been found to show signiﬁcant antitumor activities

against CCRF-HSB-2 cell line. Among them, the com-

pounds 2a,h, 3b, and 4b indicated better activities of less

than 2.0 lg/mL (IC₅₀). Against KB cell line they exhib-

ited good growth inhibitory activities of about one-tenth

antitumor potency of Ara-C (IC₅₀: 0.23 lg/mL), that is,

the IC₅₀of compounds 2a,h, 3b, and 4b were 1.97, 1.73,

3.96, and 1.00 lg/mL, respectively. The 2-deoxo-5-

deazaﬂavins may reveal fairly good and less toxic antitu-

mor activities in comparison with Ara-C. Moreover,

compounds 2b,f,i,j, 6, and 7h,l,n exhibited reasonable

potential growth inhibitory activities of ca. 5.0–7.0 lg/

mL (IC₅₀) against CCRF-HSB-2 cell line. Also com-

pounds 2b,f, 6, and 7q exhibited prospective growth

inhibitory activities of ca. 5.0–8.0 lg/mL (IC₅₀) against

KB cell line.

2.2. In vitro antitumor activities of 5-deazaﬂavins and

their 2-(N-substituted amino) derivatives against human

tumor cell lines

The compounds (2–5 and 7) synthesized in this study

were tested in vitro for their growth inhibitory activities

against two human cultured tumor cell lines, that is, hu-

On the other hand, the anti-herpes simplex virus activi-

ties of these ﬂavin analogs were investigated in vitro

against HSV-1 and HSV-2 according to the known pro-

cedure,^21,22using acyclovir as a positive control. The

potency of antiviral activity of each compound is ex-

pressed as a minimum inhibitory concentration required

to reduce virus plaque formation by 50% under experi-

mental conditions (ED₅₀). The 2-deoxo-5-deazaﬂavin

derivatives, namely 2a,b,h,j, and 4b, exhibited some

antiviral activities of 0.8, 20, 4, 20, and 4 lg/mL

(ED₅₀), respectively, against HSV-1. The obvious antivi-

ral eﬀects are not their actual antiviral potencies but

mainly attributed to the pronounced cytotoxicities of

these compounds.

Figure 2. UV–vis spectra of 6-(N-methylanilino)-2-methylthiopyrimi-

din-4(3H)-one (1a), 2-deoxo-10-methyl-2-methylthio-5-deazaﬂavin

(2a), 10-methyl-5-deazaﬂavin (5a), and 2-deoxo-2-[N-(2-hydroxyeth-

yl)-N-methylamino]-10-methyl-5-deazaﬂavin (7a).

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6341

Table 1. Growth inhibitory activities of 2-methylthio-, 2-(substituted amino)-, and 2-amino-10-alkyl-2-deoxo-5-deazaﬂavins (2–4, 6, and 7) and

5-deazaﬂavin (5) against CCRF-HSB-2 and KB tumor cell lines

Compound

Inhibitorty activity

[IC₅₀(lg/mL)]

Compound

Inhibitorty activity

[IC₅₀(lg/mL)]

Compound

Inhibitorty activity

[IC₅₀(lg/mL)]

CCRF-HSB-2

KB

CCRF-HSB-2

KB

CCRF-HSB-2

KB

68.1

8.44

2a

2b

2c

2d

2e

2f

1.91

7.07

9.57

15.3

9.27

5.92

9.87

1.82

7.06

4.84

22.6

1.96

35.6

1.97

5.42

9.27

3d

3e

4a

4b

4c

5c

6

46.4

16.4

13.7

1.64

14.3

37.1

6.04

34.9

26.3

9.5

58.5

24.8

17.9

1.0

7g

7h

>100.0

5.9

7i

24.5

25.2

27.7

7.4

38.3

32.7

50.8

16.6

82.6

8.8

12.0

7j

9.82

7.69

44.5

10.0

7k

71

2g

2h

2i

21.5

1.73

11.4

8.75

20.1

3.96

23.6

7.31

7m

7n

69.4

6.0

7a

7b

7c

7d

7e

7f

34.3

23.5

25.1

36.0

32.0

52.7

7o

7p

12.6

11.3

8.9

27.0

14.0

7.9

2j

3a

3b

3c

39.0

24.7

96.1

7q

Ara-C

0.047

0.23

The previously reported potent 2-deoxo-2-phenyl-5-

deazaﬂavins,¹which revealed minimum IC₅₀in the

range of 0.15–0.68 and 0.16–0.72 lg/mL against

CCRF-HSB-2 and KB cell lines, respectively, exhibited

ca. 3–10 times more eﬃcient antitumor activities than

those of the aforementioned 2-methylthio-, 2-amino-

and 2-(substituted amino)-2-deoxo-5-deazaﬂavins (2–4

and 7), which revealed the minimum IC₅₀in the range

of 1.64–1.96 and 1.00–1.97 lg/mL against the same cell

lines. This can be explained that the phenyl group at

the 2-position provided better aﬃnity to the enzyme

on account of the force of electrostatic attraction be-

tween the planar phenyl and the target site pocket of

the tumor cells. Hence, the phenyl derivatives exhibit a

good ﬁtting within the active site and better antitumor

activity. The SAR revealed that the highest antitumor

activities (ca. 2.0 lg/mL) for 2-deoxo-5-deazaﬂavins

were obtained with the structural features on the pyrim-

idoquinoline skeleton; SMe, N(Me)₂, or NH₂group at

the C-2 position, Et group at the N-10 position, and

Me group at the 7-position, and the moderate antitumor

activities (ca. 5.0 lg/mL) were obtained with the struc-

tural features: NHC₄H₉, morpholino or 4-methylpiper-

azinyl substituent at the C-2 position, Me group at the

N-10 position, and 7-OMe, 6,9-(OMe)₂, or 7,8-(Me)₂

substituent on the benzene ring. However, the interac-

tion of electrostatic van der Waals attraction for these

substituents with the active site of enzyme is less than

planar phenyl substituent. Therefore, they showed less

potential aﬃnities and less antitumor activities than

the 2-phenyl derivatives. It is noteworthy that the 2-ami-

no and 2-methylthio derivatives (2–4 and 7) exhibited

promising antitumor potencies in comparison with the

less potent 2,4-dioxo-5-deazaﬂavins, which revealed

lower activity (IC₅₀> 10 lg/mL) against KB cell line.²³

proliferative diseases. Therefore, PTK inhibitors can

be applied aptly as a new mode of cancer therapy.

Depending on the above-mentioned idea, herein we

investigated the AutoDock binding aﬃnities of the syn-

thesized 2-deoxo-5-deazaﬂavins (2–7) and computation-

ally designed 2-deoxo-5-deazaﬂavins (8a,b) connected

with amino acid at the 2-position into PTK. Toward

optimization of the aforementioned lead compounds

of the promising antitumor activities, the advanced

docking program AutoDock 3.05²⁴was used to evaluate

the binding free energies as potential inhibitors into the

target PTK macromolecule.

2.3.1. Validation of the accuracy and performance of

AutoDock. The most straightforward method for valida-

tion of the used scoring function is to inspect how clo-

sely the best-docked conformation resembles the

bound ligand in the experimental crystal structure. As

cited in the literature,¹⁰if the RMSD (root mean square

˚

deviation) of the best docked conformation is 62.0 A

from the experimental one, the used scoring function

is successful. Therefore, the validation of the function

implemented in AutoDock was done by docking of the

native ligand into its binding site. The docked results

were compared to the crystal structure of the bound li-

gand–protein complex. The obtained success rates of

AutoDock²⁴were highly excellent as shown in Figure

3. The STI-571 ligand (Imatinib or Gleevec), 4-(4-meth-

ylpiperazin-1-ylmethyl)-n-[4-methyl-3-(4-pyridin-3-yl-

pyrimidin-2-ylamino)phenyl]-benzamide, was docked

into its c-Kit receptor PTK (pdb code: 1t46).

˚

The RMSD of the docked ligand was 0.25 A as it seems

exactly superimposed on the native bound one. More-

over, the obtained binding free energy (DG_b) was quite

low being ꢀ18.43 kcal/mol. The docked ligand (yellow

stick) exhibited hydrogen bonds with almost same

atoms of amino acids involved with the native ligand

(ball and stick, colored by element). The docked STI li-

gand exhibited three hydrogen bonds between Cys 673,

Thr 670, and Asp 810 amino acids. Whereas, the native

ligand exhibited four hydrogen bonds between same

three amino acids involved in the docked ligand and

2.3. Molecular docking study

Both pharmaceutical companies and university labora-

tories have been active to develop compounds which

can inhibit tyrosine kinase activity in the expectation

that the potent and selective inhibitors would represent

a new class of therapeutics for cancers as well as other

6342

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

Figure 3. The docked STI ligand into PTK seems superimposed on the native STI ligand (ball and stick, colored by elements). The docked ligand

(yellow stick) exhibited hydrogen bonds (green dotted lines) with almost same amino acids involved by the native one, the DG_b: ꢀ18.43 kcal/mol, and

˚

RMSD: 0.25 A.

another amino acid of Glu 640. These results indicated

the high accuracy of the AutoDock simulation in com-

parison with the biological methods.²⁵

and Tyr 672 and the terminal hydrocarbon chain of

˚

Leu 595 within distance of 3.46, 4.53, and 3.36 A,

respectively. Also the 2-methylthio group interacts with

lys 623, Val 668, and Cys 809 by hydrophobic attraction

˚

within distance of 3.22, 3.90, and 3.47 A, respectively.

The interactions keep these derivatives in the binding

site, providing better RMSD values than other deriva-

˚

tives (3.80–5.41 A). Exceptionally, compounds 2c and

2 e showed one hydrogen bond between the C₄-oxo

and HO of Thr 670. This aforementioned docking

results of 2a–j indicate that these compounds are not

expected to be a reasonable candidate for PTK

inhibition.

2.3.2. AutoDock binding aﬃnities of the synthesized and

the designed compounds into PTK. The binding aﬃnity

was evaluated by the binding free energies (DG_b, kcal/

mol), inhibition constants (K_i), hydrogen bonding, and

RMSD values. The compounds which revealed the high-

est binding aﬃnities, that is, lowest binding free ener-

gies, within PTK and the hydrogen bond interactions

into the target macromolecule are represented in Table

2. These compounds include 7-substituted 2-deoxo-10-

methyl-2-dimethylamino-5-deazaﬂavins (3d,e), 10-alkyl-

2-amino-2-deoxo-5-deazaﬂavins (4a–c), 2-(substituted

The molecular docking study revealed that compounds

8a,b prepared by replacement of 2-methylthio moiety

of 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2) by

amino acids such as glycine and histidine exhibited rea-

sonable binding energies and more important higher

number of hydrogen bonds. The comparative docking

modes of 8a,b and 7n into c-Kit receptor PTK with its

bound ligand STI are shown in detail in Figure 4. The

amino acid coupled to 5-deazaﬂavins has been shown

to enhance remarkably the binding potentials in PTK.

The higher aﬃnity of these designed derivatives is pre-

sumably attributed to the formation of more hydrogen

bonds (4–6 hydrogen bonds) and/or tighter hydrogen

bonds between their 2-amino acid moieties and the bind-

ing site. While 10-alkyl-2-deoxo-2-methylthio-5-deazaf-

lavins (2) and other 2-(substituted amino)-2-deoxo-5-

deazaﬂavins (3, 6, and 7) exhibited less binding aﬃnities

into the binding sites of PTK. This may be explained by

missing of substituents at C₂-position which provide

tight hydrogen bond interaction or surface electrostatic

attraction such as 2-phenyl group as cited in our previ-

ous publication.¹

amino)-10-alkyl-2-deoxo-5-deazaﬂavins

(7c,e,n,p,q),

and the computationally designed 2-deoxo-5-deazaﬂav-

ins connected with amino acids (8a,b). The latter deriv-

atives exhibited many hydrogen bonds (4–6) between the

target PTK. As shown in Table 2, compound 8a (DG_b: –

8.88 kcal/mol) exhibited four hydrogen bonds with Thr

˚

670 and RMSD: 5.67 A, compound 8b, (DG_b:

ꢀ10.63 kcal/mol) exhibited six hydrogen bonds with

Lys 623, Val 668, Thr 670, and Asp 810 and RMSD:

˚

4.29 A, compound 7c (DG_b: ꢀ13.37 kcal/mol) exhibited

two hydrogen bonds with Ala 621 and Thr 670 and

˚

RMSD: 4.78 A, compound 7n (DG_b: ꢀ14.50 kcal/mol)

exhibited one hydrogen bond with Cys 673 and RMSD:

˚

7.93 A, and compound 7q (DG_b: ꢀ15.05 kcal/mol)

exhibited one hydrogen bond with Thr 670 and RMSD:

˚

3.80 A.

To investigate the potential PTK inhibition, the compar-

atively antitumor active 10-alkyl-2-deoxo-2-methylthio-

5-deazaﬂavins (2a–j) were also docked into PTK

(1t46). Unfortunately, the docking of most compounds

˚

(2) was too far for the distance (>2.50 A) between the

C₄-oxo group and HO of Thr 670 and/or HN of Lys

623 (as common hydrogen bond donors) to form the

hydrogen bond into the binding site. However, the pla-

nar pyrimidoquinoline ring was involved in hydropho-

bic electrostatic surface interaction, where it was

sandwiched between the phenyl moieties of Phe 811

Figure 5 illustrates docking of compound 4b (DG_b:

ꢀ12.16 kcal/mol) in another region of the binding site

along with STI-ligand and 4b exhibited one hydrogen

bond. Whereas, compounds 7c and 7q were docked in

another groove with two and one hydrogen bonds,

respectively. This diﬀerent binding mode of these com-

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6343

Table 2. The best docking results based on the binding free energies (DG_b) and inhibition constants (K_i) of compounds docked into PTK, the

distances and angles of hydrogen bonds between compounds and amino acids involved in PTK, and RMSD from the crystallized STI ligand

a

c

RMSD (A)

K_i^b

Hydrogen bonds between atoms of compounds and amino acids

˚

Compound

DG_b(kcal/mol)

Atom of compound

Amino acid

Distance (A)

Angle (°)

3d

3e

ꢀ12.23

ꢀ12.87

1.09E-09

3.66E-10

C₄-Oxo

HN of Cys673

HN of Thr670

HO of Thr670

2.34

2.40

1.88

150.0

127.1

153.6

9.37

3.73

4a

ꢀ12.71

4.82E-10

C₂-NH_a

C₂-NH_b

N of Lys623

N of Thr670

1.91

2.49

163.1

153.0

3.02

4b

4c

ꢀ12.16

ꢀ12.97

1.21E-09

3.11E-10

C₂-NH

OH of Glu640

O of Thr670

1.91

1.96

120.2

139.6

4.61

6.19

7c

ꢀ13.37

1.59E-10

C₂-OH

C₂-O

O of Ala 621

HO of Thr670

2.45

1.72

121.8

140.1

4.78

d

7e

7n

7p

ꢀ14.27

ꢀ14.50

ꢀ13.47

3.49E-11

1.59E-10

1.35E-10

C₄-Oxo

C₂-OH

C₂-O

—

6.51

7.93

5.73

HN of Cys673

OH of Thr670

HO of Thr670

2.15

2.13

2.04

124.0

143.1

144.9

7q

8a

ꢀ15.05

ꢀ8.88

9.34E-12

3.11E-07

C₂-NH

COOH

OH of Thr670

2.03

2.07

2.37

157.2

131.8

139.2

3.80

5.67

COOH

COO

C₂-NH

CO

N of Thr670

2.24

2.23

2.11

2.35

127.7

147.0

121.3

119.5

HO of Thr670

OH of Thr670

HNl of Thr670

8b

ꢀ10.63

ꢀ18.43

1.63E-08

3.08E-14

COO

COOH

HN of Lys623

O of Val668

1.33

1.71

2.01

2.18

1.66

128.1

131.2

148.6

121.5

161.0

4.29

0.25

CO

NH (imidazole)

N3

HO of Thr670

O of Asp 810

HN of Cys673

STI^e

NH (H79)

029

OH of Thr670

HN of Asp 810

1.86

2.01

147.2

133.3

^aBinding free energy.

^bInhibition constant.

^cRoot mean square deviation.

^dThe angle of the detected H-bond (91.8°) does not ﬁt with the angle requirements.

^eThe native co-crystalline ligand (STI-571) bound to PTK (PDB code: It46).

pounds may explain their diﬀerent antiproliferative

activity against CCRF-HSB-2 and KB tumor cell lines

as cited in Table 1.

The overall correlation between the growth inhibitory

activities (IC₅₀, lg/mL) of the synthesized 5-deazaﬂavins

against tumor cells and the binding aﬃnities predicted

Figure 4. Diﬀerential binding aﬃnities of the synthesized 2-deoxo-5-deazaﬂavin (7n; indigo, stick) and the designed 2-deoxo-5-deazaﬂavins connected

with amino acids (8a: yellow, ball and stick; 8b: colored by element stick) into PTK. Compounds 8a,b exhibited the higher binding with 4–6 hydrogen

bonds. The binding pocket of PTK is shown in transparent solid surface with labeled amino acids and the STI ligand is shown as red line. The settled

hydrogen bonds are shown as green lines.

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H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

Figure 5. Inhibitor 4b (colored by element, ball and stick) is docked horizontally into the plane of the groove of the binding site of PTK, where its C₂-

NH₂group was involved in hydrogen bond with Glu 640, while 7c (colored by elememt, stick) is embedded perpendicularly into the groove exhibiting

two hydrogen bonds. Compound 7q (yellow, stick) is extended through the whole groove of the pocket forming one hydrogen bond with Thr 670.

The binding pocket is shown in solid surface and the STI ligand is shown as red line.

Whereas, the growth inhibition against KB cells re-

vealed a reasonable correlation with AutoDock binding

free energies for compounds 3c,d, 4c, and 7a–e,h,i,l of

correlation coeﬃcient (R²) value of 0.599 as shown in

Figure 7.

3. Conclusions

In this study, various novel 10-alkyl-2-deoxo-2-methyl-

thio-5-deazaﬂavins (2a–j) were synthesized from 6-(N-

monoalkylanilino)-2-methylthiopyrimidin-4(3H)-ones

(1a–j) by the reaction using Vilsmeier reagent. The sim-

ilar 2-(substituted amino)-10-alkyl-2-deoxo-5-deazaﬂav-

ins (3a–e, 6, and 7a–q) were synthesized by the facile

replacement of C₂-methylthio moiety by amines of dif-

ferent type. 10-Alkyl-2-amino-2-deoxo-5-deazaﬂavins

(4a–c) were also prepared in better yield than the pub-

lished method for compounds 4a,b. Additionally, 5-

deazaﬂavins {pyrimido[4,5-b]quinolin-2,4-diones} (5a–

c) were prepared by acidic hydrolysis of 2-deoxo-2-

methylthio-5-deazaﬂavins (2) in good yields. In the

growth inhibitory activities of 2–7 against T-cell acute

lymphoblastoid leukemia cell line (CCRF-HSB-2) and

human oral epidermoid carcinoma cell line (KB)

in vitro, many derivatives showed potential antitumor

activities. Among them, compounds 2a,h, 3b, and 4b

exhibited the most signiﬁcant antiproliferative potencies

with IC₅₀of 1.64–1.96 and 1.00–3.96 lg/mL against

CCRF-HSB-2 and KB cells, respectively. Some other

derivatives, namely 2b,f,i,j, 6, and 7h,m,p showed IC₅₀

of 4.84–7.40 and 5.42–7.90 lg/mL against CCRF-HSB-

2 and KB cells, respectively. These results revealed a

promising antitumor activity of many derivatives, which

are considered as new leads.

Figure 6. Correlation between the binding free energy (DG_b) and IC₅₀

of 2-(substituted amino)-10-alkyl-2-deoxo-5-deazaﬂavins (3a,c,d and

7a,b,d,e,h–l,o) against human T-cell acute lymphoblastoid leukemia

cell line (CCRF-HSB-2).

Figure 7. Correlation between the binding free energy (DG_b) and IC₅₀

of 2-amino- and 2-(substituted amino)-10-alkyl-2-deoxo-5-deazaﬂavins

(3c,d,4c, and 7a–e,h,i,l) against human oral epidermoid carcinoma cell

line (KB).

by AutoDock was fairly good for some compounds.

Considering the growth inhibition against CCRF-

HSB-2 cells, it was noticed that the correlation between

IC₅₀of 3a,c,d and 7a,b,d,e,h–l,o and their AutoDock

binding free energies revealed an excellent correlation

coeﬃcient (R²) of 0.809 as represented in Figure 6.

The AutoDock investigation of the synthesized 2-deoxo-

5-deazaﬂavins (2–4, 6, and 7) and the computationally

designed 2-deoxo-5-deazaﬂavins connected with amino

acids (8a,b) was carried out for lead optimization. Thus,

they were docked within c-kit protein tyrosine kinase.

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6345

The overall correlation between the growth inhibitory

activities (IC₅₀, lg/mL) of the synthesized 5-deazaﬂavins

against tumor cells and the binding aﬃnities predicted

by AutoDock was fairly good for some compounds,

namely, 3a,c,d and 7a,b,d,e,h–l,o against CCRF-HSB-2

cell line, with the correlation coeﬃcient (R²) of 0.809.

While the correlation between IC₅₀of compounds

3c,d, 4c, and 7a–e,h,i,l against KB cell line was also good

with the correlation coeﬃcient (R²) of 0.599. Further, in

order to enhance the binding aﬃnity of these deriva-

tives, 5-deazaﬂavins connected with amino acids were

designed computationally. They exhibited preferential

binding aﬃnities into PTK with more hydrogen bonds

and lower binding free energies. These computationally

designed hybrid compounds may be promising candi-

dates for further antitumor investigation and their syn-

theses are in progress.

EtOH); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

241 (4.28), 263sh (4.15), 292sh (3.89); IR (m_max/cm^ꢀ1):

3220 (NH), 1640 (CO); H NMR (CDCl₃): d 2.46 (3H,

s, 2-SMe), 3.45 (3H, s, 6-NMe), 5.06 (1H, s, 5-H),

7.11–7.30 (3H, m, Ph-o,pH), 7.36–7.43 (2H, m, Ph-

mH), 13.24 (1H, br s, 3-NH, exchangeable with D₂O);

Anal. calcd for C₁₂H₁₃N₃OS: C, 58.28; H, 5.30; N,

16.99. Found C, 58.23; H, 5.43; N, 16.95.

1

4.1.3. 6-(N-Ethylanilino)-2-methylthiopyrimidin-4(3H)-

one (1b). Yield, (2.89 g, 65%); mp 235–236 °C (from

EtOH); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

241 (4.56), 261sh (4.40), 293sh (4.10) ; IR (m_max/cm^ꢀ1):

1

3200 NH), 1640 (CO); H NMR (CDCl₃): d 1.20 (3H,

t, J = 6.9 Hz, 6-NCH₂-CH₃), 2.48 (3H, s, 2-SMe), 3.94

(2H, q, J = 6.9 Hz, 6-NCH₂-CH₃), 4.90 (1H, s, 5-H),

7.15–7.22 (2H, m, Ph-oH), 7.28–7.34 (2H, m, Ph–pH),

7.37–7.45 (2H, m, Ph–mH), 13.35 (1H, br s, 3-NH,

exchangeable with D₂O); Anal. calcd for C₁₃H₁₅N₃OS:

C, 59.74; H, 5.79; N, 16.08. Found: C, 59.58; H, 5.81;

N, 16.10.

4. Experimental

4.1. Chemistry

4.1.4. 6-(N-Methyl-4-methylanilino)-2-methylthiopyrimi-

din-4(3H)-one (1c). Yield, (3.51 g, 79%); mp 301–303 °C

Mps were obtained on a Yanagimoto micro melting

point apparatus and are uncorrected. Microanalyses

were measured by Yanaco CHN Corder MT-5 appara-

tus. IR spectra were recorded on a JASCO FT/IR-200

spectrophotometer as Nujol mulls. ¹H NMR spectra

were obtained using a Varian VXR 300 MHz spectro-

photometer and chemical shift values were expressed

in d values (ppm) relative to tetramethylsilane (TMS)

as internal standard. Coupling constants are given in

Hz. All NH and OH protons were exchangeable with

D₂O. UV spectra were measured in absolute EtOH

using Beckman DU-68S UV spectrophotometer and

the wavelength value with sh refers to wavelength at

which shoulder or inﬂexion occurs in the absorption.

All reagents were of commercial quality and were used

without further puriﬁcation. Organic solvents were

dried in the presence of an appropriate drying agent

and were stored over suitable molecular sieves. Reaction

progress was monitored by analytical thin layer chro-

matography (TLC) on pre-coated glass plates (silica

gel 60F₂₅₄-plate-Merck) and the products were visual-

ized by UV light.

(from EtOH); UV (EtOH):

k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 242 (4.56), 259sh (4.47), 292sh

(4.16); IR (m_max/cm^ꢀ1): 3200 (NH), 1625 (CO); ¹H

NMR (CDCl₃): d 2.36 (3H, s, pMe), 2.48 (3H, s, 2-

SMe), 3.43 (3H, s, 6-N-Me), 5.02 (1H, s, 5-H), 7.09

0

(2H, d, J_{2 ;3}¼ J_{5 ;6}¼ 8:1 Hz, Ar-oH), 7.19 (2H, d,

0

J_{2 ;3}¼ J_{5 ;6}¼ 8:1 Hz, Ar-mH), 12.94 (1H, br s, 3-NH,

exchangeable with D₂O); Anal. calcd for C₁₃H₁₅N₃OS:

C, 59.74; H, 5.79; N, 16.08 . Found: C, 59.66; H, 5.70;

N, 15.86.

4.1.5. 6-(N-Methyl-2-methylanilino)-2-methylthiopyrimi-

din-4(3H)-one (1d). Yield, (2.35 g, 53%); mp 246–248 °C

(from MeOH); UV (EtOH):

k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 240 (4.59), 267sh (4.33), 290sh

(4.09); IR (m_max/cm^ꢀ1): 3200 (NH), 1620 (CO); ¹H

NMR (CDCl₃): d 2.15 (3H, s, oMe), 2.57 (3H, br s, 2-

SMe), 3.39 (3H, s, 6-NMe), 4.62 (1H, br s, 5-H), 7.08–

7.13 (1H, m, Ar-oH), 7.21–7.25 (3H, m, Ar-m,pH),

13.07 (1H, br s, 3-NH, exchangeable with D₂O); Anal.

calcd for C₁₃H₁₅N₃OS: C, 59.74; H, 5.74; N, 16.08.

Found: C, 59.72; H, 5.97; N, 15.85.

4.1.1. General procedure for the preparation of 6-(N-

alkylanilino)-2-methylthiopyrimidin-4(3H)-ones (1a–j). A

4.1.6. 6-(N-Methyl-4-methoxyanilino)-2-methylthiopyr-

imidin-4(3H)-one (1e). Yield, (3.58 g, 76%); mp 292–

293 °C (from EtOH); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 246 (4.44), 275sh (4.20), 289sh

(4.07); IR (m_max/cm^ꢀ1): 3200 (NH), 1630 (CO); ¹H

NMR (CDCl₃): d 2.48 (3H, s, 2-SMe), 3.42 (3H, s, 6-

NMe), 3.82 (3H, s, pOMe), 4.98 (1 H, s, 5-H), 6.91 (2

mixture

of

6-chloro-2-methylthiopyrimidin-4(3H)-

one^14,16(3.0 g, 0.017 mol) and an appropriate N-alky-

laniline (0.051–0.085 mol) in n-butanol (20 mL) was

reﬂuxed with stirring for 12–72 h. After cooling, the pre-

cipitated solid was ﬁltered oﬀ to get the ﬁrst crop. The

ﬁltrate was concentrated in vacuo and the residue was

crushed with diethyl ether to precipitate powdery crys-

tals, which were ﬁltered oﬀ, washed with water, and

dried to aﬀord the second crop. The collected solids were

crystallized from appropriate solvents with charcoal to

aﬀord the corresponding products as colorless needles

in 55–83% yields.

0

H, d, J_{2 ;3}¼ J_{5 ;6}¼ 9:0 Hz, Ar-oH), 7.12 (2H, d,

0

J_{2 ;3}¼ J_{5 ;6}¼ 9:0 Hz, Ar-mH), 12.78 (1H, br s, 3-NH,

exchangeable with D₂O); Anal. calcd for C₁₃H₁₅N₃O₂S:

C, 56.30; H, 5.45; N, 15.15. Found: C, 56.13; H, 5.44; N,

15.30.

4.1.7. 6-(N-Methyl-3-methoxyanilino)-2-methylthiopyr-

imidin-4(3H)-one (1f). Yield, (3.54 g, 75%); mp 196–

198 °C (from EtOH); UV (EtOH): k_max/nm (log e/

4.1.2. 6-(N-Methylanilino)-2-methylthiopyrimidin-4(3H)-

one (1a). Yield, 3.11 g (74%); mp 268–270 °C (from

6346

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

dm³mol^ꢀ1cm^ꢀ1): 240 (4.56), 261sh (4.42), 287sh (4.20);

IR (m_max/cm^ꢀ1): 3200 (NH), 1635 (CO); ¹H NMR

(CDCl₃): d 2.48 (3H, s, 2-SMe), 3.44 (3H, s, 6-NMe),

3.80 (3H, s, mOMe), 5.08 (1H, s, 5-H), 6.73–6.77 (1H,

m, Ar-pH), 6.78–6.85 (2H, m, Ar-oH), 7.30 (1H, t,

4.1.12. General procedure for the preparation of 10-alkyl-

2-deoxo-2-methylthio-5-deazaﬂavins {10-alkyl-2-methyl-

thiopyrimido[4,5-b]quinolin-4(10H)-ones} (2a–j). A mix-

ture of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-

ones (1, 0.01 mol) and phosphoryl chloride (7.7 g,

0.05 mol) in anhydrous dimethylformamide (10 mL)

was heated under stirring at 90 °C for 0.5–2 h. Then, the

reaction mixture was poured onto ice and neutralized

with aqueous ammonia (pH 7). The yellow crystals

separated were ﬁltered oﬀ, washed with water, dried,

and recrystallized from an appropriate solvent to aﬀord

the corresponding product as yellow needles in 71–99%

yields.

0

J_{4 ;5}¼ J_{5 ;6}¼ 8:4 Hz, Ar-mH), 13.02 (1H, br s, 3-NH,

exchangeable with D₂O); Anal. calcd for C₁₃H₁₅N₃O₂S:

C, 56.30; H, 5.45; N, 15.15. Found: C, 56.39; H, 5.48; N,

14.90.

4.1.8. 6-(N-Methyl-4-chloroanilino)-2-methylthiopyrimi-

din-4(3H)-one (1g). Yield, (3.98 g, 83%); mp >300 °C

(from MeOH); UV (EtOH):

k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 239 (4.51), 262sh (4.43), 291sh (4.21);

IR (m_max/cm^ꢀ1): 3200 (NH), 1620 (CO); ¹H NMR

(CDCl₃): d 2.44 (3H, s, 2-SMe), 3.41 (3H, s, 6-NMe),

4.1.13. 10-Methyl-2-methylthiopyrimido[4,5-b]quinolin-

4(10H)-one (2a). Yield, (2.55 g, 99%); mp 281–282 °C

(decomp. from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 222 (4.57), 268 (4.24), 286 (4.42),

0

5.07 (1H, s, 5-H), 6.91 (2H, d, J_{2 ;3}¼ J_{5 ;6}¼ 8:7 Hz, Ar-

0

oH), 7.12 (2H, d, J_{2 ;3}¼ J_{5 ;6}¼ 8:7 Hz, Ar-mH), 11.89

(1H, br s, 3-NH, exchangeable with D₂O); Anal. calcd

for C₁₂H₁₂ClN₃OS: C, 51.15; H, 4.29; N, 14.91. Found:

C, 50.85; H, 4.28; N, 14.88.

1

340 (4.12), 423 (4.17); IR (m_max/cm^ꢀ1): 1655 (CO); H

NMR (CDCl₃): d 2.62 (3H, s, 2-SMe), 4.29 (3H, s, 10-

Me), 7.60 (1H, t, J_6,7= J_7,8= 8.7 Hz, 7-H), 7.81 (1H,

d, J_8,9= 8.7 Hz, 9-H), 7.97 (1H, t, J_7,8= J_8,9= 8.7 Hz,

8-H), 7.02 (1H, d, J_6,7= 8.7 Hz, 6-H), 9.24 (1H, s, 5-

H); Anal. calcd for C₁₃H₁₁N₃OS: C, 60.68; H, 4.31; N,

16.33. Found: C, 60.30; H, 4.53; N, 15.94.

4.1.9. 6-(N-Ethyl-4-methylanilino)-2-methylthiopyrimi-

din-4(3H)-one (1h). Yield, (2.86 g, 61%); mp 237–

239 °C (from EtOH); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 242 (4.63), 260sh (4.48), 292sh

(4.16); IR (m_max/cm^ꢀ1): 3200 (NH), 1635 (CO); ¹H

NMR (CDCl₃): d 1.19 (3H, t, J = 7.2 Hz, 6-NCH₂-

CH₃), 2.37 (3H, s, pMe), 2.49 (3H, s, 2-SMe), 3.91

(2H, q, J = 7.2 Hz, 6-NCH₂-CH₃), 4.86 (1H, s, 5-H),

4.1.14.

10-Ethyl-2-methylthiopyrimido[4,5-b]quinolin-

4(10H)-one (2b). Yield, (2.69 g, 99%); mp 262–265 °C (de-

comp. from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 222 (4.64), 268sh (4.32), 286 (4.49),

1

7.04 (2H, d, J_{2 ;3}¼ J_{5 ;6}¼ 8:4 Hz, Ar-oH), 7.19 (2H,

338 (4.19), 424 (4.25); IR (m_max/cm^ꢀ1): 1655 (CO); H

0

d, J_{2 ;3}¼ J_{5 ;6}¼ 8:4 Hz, Ar-mH), 13.13 (1H, br s, 3-

NH, exchangeable with D₂O); Anal. calcd for

C₁₄H₁₇N₃OS: C, 61.06; H, 6.22; N, 15.26. Found: C,

61.12; H, 6.24; N, 15.50.

NMR (CDCl₃): d 1.54 (3H, t, J = 6.9 Hz, 10-CH₂-CH₃),

2.63 (3H, s, 2-SMe), 4.96 (2H, q, J = 6.9 Hz, 10-CH₂-

CH₃), 7.59 (1H, dt,

1

H, J_6,7= J_7,8= 9.0 Hz,

J_7,9= 1.5 Hz, 7-H), 7.82 (1H, d, J_8,9= 9.0 Hz, 9-H), 7.97

(1H, dt, J_7,8= J_8,9= 9.0 Hz, J_6,8= 1.5 Hz, 8-H), 8.04

(1H, dd, J_6,7= 9.0 Hz, J_6,8= 1.5 Hz, 6-H), 9.24 (1H, s,

5-H); Anal. calcd for C₁₄H₁₃N₃OS: C, 61.97; H, 4.83; N,

15.49. Found: C, 62.08; H, 5.01; N, 15.12.

4.1.10. 6-(N-Methyl-3,4-dimethylanilino)-2-methylthio-

pyrimidin-4(3H)-one (1i). Yield, (3.65 g, 78%); mp 281–

283 °C (from EtOH); UV (EtOH): k_max/nm (loge/

dm³mol^ꢀ1cm^ꢀ1): 243 (4.55), 260sh (4.44), 292sh

(4.13); IR (m_max/cm^ꢀ1): 3200 (NH), 1625 (CO); ¹H

NMR (CDCl₃): d 2.25 (3H, s, pMe), 2.26 (3H, s,

mMe), 2.49 (3H, s, 2-SMe), 3.42 (3H, s, 6-NMe), 5.00

4.1.15. 7,10-Dimethyl-2-methylthiopyrimido[4,5-b]quino-

lin-4(10H)-one (2c). Yield, (2.66 g, 98%); mp 279–281 °C

(decomp. from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 225 (4.62), 270 (4.36), 288 (4.46),

0

(1H, s, 5-H), 6.92 (1H, dd, J_{5 ;6}¼ 7:8 Hz,

1

J_{2 ;6}¼ 1:5 Hz , N-Ar-6⁰H), 6.98 (1H, d, J_{2 ;6}¼ 1:5 Hz,

340 (4.19), 432 (4.23); IR (m_max/cm^ꢀ1): 1640 (CO); H

0

Ar-2⁰H), 7.14 (1H, d, J_{5 ;6}¼ 7:8 Hz, Ar-5⁰H), 12.74

(1H, br s, 3-NH, exchangeable with D₂O); Anal. calcd

for C₁₄H₁₇N₃OS: C, 61.06; H, 6.22; N, 15.26. Found:

C, 61.13; H, 6.17; N, 14.95.

NMR (CDCl₃): d 2.56 (3H, s, 7-Me), 2.62 (3H, s, 2-

SMe), 4.28 (3H, s, 10-Me), 7.71 (1H, d, J_8,9= 8.7 Hz,

9-H), 7.75–7.80 (1H, m, 8-H), 7.81 (1H, d,

J_6,8= 2.1 Hz, 6-H), 9.16(1H, s, 5-H); Anal. calcd for

C₁₄H₁₃N₃OS: C, 61.97; H, 4.83; N, 15.49. Found: C,

61.66; H, 4.89; N, 15.27.

0

4.1.11. 6-(N-Methyl-2,5-dimethoxyanilino)-2-methylthio-

pyrimidin-4(3H)-one (1j). Yield, (3.76 g, 72%); mp 227–

229 °C (from EtOH); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 240 (4.50), 266sh (4.23), 292sh

(4.16); IR (m_max/cm^ꢀ1): 3200 (NH), 1630 (CO); ¹H

NMR (CDCl₃): d 2.50 (3H, br s, 2-SMe), 3.36 (3H, s,

6-NMe), 3.75 (3H, s, 2⁰-OMe), 3.76 (3H, s, 5⁰-OMe),

4.1.16. 9,10-Dimethyl-2-methylthiopyrimido[4,5-b]quino-

lin-4(10H)-one (2d). Yield, (1.93 g, 71%); mp 273–274 °C

(decomp. from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 227 (4.56), 270 (4.36), 290 (4.52),

1

351 (4.26), 429 (4.21); IR (m_max/cm^ꢀ1): 1650 (CO); H

4.87 (1H, s, 5-H), 6.73 (1H, d, J_{4 ;6}¼ 3:0 Hz, Ar-6⁰-

NMR (CDCl₃): d 2.63 (3H, s, 2-SMe), 2.91 (3H, s, 9-

Me), 4.35 (3H, s, 10-Me), 7.45 (1H, t,

J_6,7= J_7,8= 7.5 Hz, 7-H), 7.70–7.76 (1H, m, 8-H), 7.82

(1H, d, J_6,7= 7.5 Hz, 6-H), 9.14 (1H, s, 5-H); Anal. calcd

for C₁₄H₁₃N₃OS: C, 61.97; H, 4.83; N, 15.49. Found: C,

61.73; H, 4.96; N, 15.10.

0

H), 6.83 (1H, dd, J_{4 ;6}¼ 9:0 Hz, J_{4 ;6}¼ 3:0 Hz, Ar-

4⁰H), 6.90 (1H, d, J_{4 ;6}¼ 9:0 Hz, Ar-3⁰H), 12.93 (1H,

br s, 3-NH, exchangeable with D₂O); Anal. calcd for

C₁₄H₁₇N₃O₃S: C, 54.71; H, 5.57; N, 13.67. Found: C,

54.81; H, 5.62; N, 13.39.

0

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6347

4.1.17. 7-Methoxy-10-methyl-2-methylthiopyrimido[4,5-

b]quinolin-4(10H)-one (2e). Yield, (2.7 g, 94%); mp

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 213 (4.31), 259 (4.26), 278

sh (4.09), 311 (4.41), 403 (4.33); IR (m_max/cm^ꢀ1): 1660

(CO); H NMR (CDCl₃): d 2.63 (3H, s, 2-SMe), 3.96

(3H, s, 9-OMe), 3.98 (3H, s, 6-OMe), 4.48 (3H, s, 10-

Me), 6.78 (1H, d, J_7,8= 9.0 Hz, 7-H), 7.31 (1H, d,

J_8,9= 9.0 Hz, 8-H), 9.63 (1H, s, 5-H); Anal. calcd for

C₁₅H₁₅N₃O₃S: C, 56.77; H, 4.76; N, 13.24. Found: C,

56.65; H, 4.78; N, 13.01.

1

275–277 °C (decomp. from DMF); UV (EtOH): k_max

/

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 229 (4.48), 275 (4.40), 290

(4.43), 339 (4.12), 450 (4.19); IR (m_max/cm^ꢀ1): 1650

1

(CO); H NMR (CDCl₃): d 2.63 (3H, s, 2-SMe), 3.97

(3H , s, 7-OMe), 4.31 (3H, s, 10-Me), 7.31 (1H, d,

J_6,8= 3.0 Hz, 6-H), 7.61 (1H, dd, J_8,9= 9.3 Hz,

J_6,8= 3.0 Hz, 8-H), 7.77 (1H, d, J_8,9= 9.3 Hz, 9-H),

9.19 (1H, s, 5-H); Anal. calcd for C₁₄H₁₃N₃O₂S: C,

58.52; H, 4.56; N, 14.62. Found: C, 58.26; H, 4.67; N,

14.22.

4.1.23. General procedure for the preparation of 10-alkyl-

2-deoxo-2-dimethylamino-5-deazaﬂavins {10-alkyl-2-dim-

ethylaminopyrimido[4,5-b]quinolin-4(10H)-ones} (3a–e).

A mixture of 10-alkyl-2-deoxo-2-methylthio-5-deazaﬂa-

vin (2, 5.0 mmol) and 50% aqueous dimethylamine

(50 mL) was heated in steel sealed tube at 135 °C

(10 kg/cm²pressure) for 4 h. After the reaction was com-

plete, the precipitated crystals were collected by ﬁltera-

tion, and mother liquid was evaporated in vacuo to

get the second crop. The product was washed with

water, dried and recrystallized from an appropriate sol-

vent to aﬀord the corresponding product as yellow crys-

tals in 59–96% yields.

4.1.18. 8-Methoxy-10-methyl-2-methylthiopyrimido[4,5-

b]quinolin-4(10H)-one (2f). Yield, (2.70 g, 94%); mp 285–

287 °C (decomp. from DMF); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 231 (4.60), 256 (4.25), 281

(4.44), 359 (4.05), 417 (4.47); IR (m_max/cm^ꢀ1): 1630 (CO);

¹H NMR (CDCl₃): d 2.60 (3H, s, 2-SMe), 4.07 (3H, s, 8-

OMe), 4.21 (3H, s, 10-Me), 7.03 (1H, d, J_7,9= 2.4 Hz, 9-

H), 7.18 (1H, dd, J_6,7= 9.0 Hz, J_7,9= 2.4 Hz, 7-H), 7.89

(1H, d, J_6,7= 9.0 Hz, 6-H), 9.09 (1H, s, 5-H); Anal. calcd

for C₁₄H₁₃N₃O₂S: C, 58.52; H, 4.56; N, 14.62. Found: C,

58.42; H, 4.63; N, 14.36.

4.1.24. 10-Methyl-2-dimethylaminopyrimido[4,5-b]quino-

lin-4(10H)-one (3a). Yield, (1.22 g, 96%); mp >300 °C

4.1.19. 7-Chloro-10-methyl-2-methylthiopyrimido[4,5-b]-

quinolin-4(10H)-one (2g). Yield, (2.86 g, 98%); mp 287–

289 °C (decomp. from DMF); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 227 (4.63), 272 (4.49), 291

(4.46), 332 (4.12), 434 (4.19); IR (m_max/cm^ꢀ1): 1650 (CO);

¹H NMR (CDCl₃): d 2.62 (3H, s, 2-SMe), 4.27 (3H, s,

10-Me), 7.75 (1H, d, J_8,9= 9.3 Hz, 9-H), 7.61 (1H, dd,

J_8,9= 9.3 Hz, J_6,8= 2.4 Hz, 8-H), 7.97 (1H, d,

J_6,8= 2.4 Hz, 6-H), 9.12 (1H, s, 5-H); Anal. calcd for

C₁₃H₁₀ClN₃OS: C, 53.52; H, 3.45; N, 14.40. Found: C,

53.55; H, 3.66; N, 14.17.

(from DMF); UV (EtOH):

k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 220 (4.65), 271 (4.68), 279sh (4.57),

320 (4.12), 382sh (4.15), 408 (4.33), 432 (4.28); IR

(m_max/cm^ꢀ1): 1655 (CO); ¹H NMR (CDCl₃): d 3.34

(6H, s, 2-N(CH₃)₂), 4.12 (3H, s,10-Me), 7.44 (1H, t,

J_6,7= J_7,8= 7.8 Hz, 7-H), 7.65 (1H, d, J_8,9= 8.7 Hz, 9-

H), 7.81 (1H, t, J_7,8= 7.8 Hz, J_8,9= 8.7 Hz, 8-H), 7.86

(1H, d, J_6,7= 7.8 Hz, 6-H), 8.94 (1H, s, 5-H); Anal.

calcd for C₁₄H₁₄N₄OÆ0.1H₂O: C, 65.66; H, 5.59; N,

21.88. Found: C, 65.55; H, 5.80; N, 21.50.

4.1.25. 10-Ethyl-2-dimethylaminopyrimido[4,5-b]quinolin-

4(10H)-one (3b). Yield, (0.79 g, 59%); mp >300 °C (from

DMF); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

220 (4.55), 271 (4.53), 278sh (4.48), 322 (3.96), 383sh

(4.04), 407 (4.22), 431 (4.16); IR (m_max/cm^ꢀ1): 1650

4.1.20. 10-Ethyl-7-methyl-2-methylthiopyrimido[4,5-b]-

quinolin-4(10H)-one (2h). Yield, (2.71 g, 95%); mp 278–

280 °C (decomp. from DMF); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 225 (4.65), 269 (4.41), 289

(4.49), 340 (4.23), 432 (4.26); IR (m_max/cm^ꢀ1): 1655

1

(CO); H NMR (CDCl₃): d 1.49 (3H, t, J = 7.2 Hz, 10-

1

(CO); H NMR (CDCl₃): d 1.52 (3H, t, J = 7.2 Hz, 10-

CH₂-CH₃), 3.34 (3H, s, 2-NMe), 3.48 (3H, s, 2-NMe),

4.79 (2H, q, J = 7.2 Hz, 10-CH₂-CH₃), 7.43 (1H, dt,

J_6,7= J_7,8= 7.8 Hz, J_7,9= 1.8 Hz, 7-H), 7.65 (1H, d,

J_8,9= 8.7 Hz, 9-H), 7.81 (1H, dt, J_7,8= 7.8 Hz,

J_8,9= 8.7 Hz, J_6,8= 1.8 Hz, 8-H), 7.90 (1 H, dd,

J_6,7= 7.8 Hz, J_6,8= 1.8 Hz, 6-H), 8.95 (1H, s, 5-H);

Anal. calcd for C₁₅H₁₆N₄O: C, 67.15; H, 6.01; N,

20.88. Found: C, 66.86; H, 6.12; N, 20.70.

CH₂-CH₃), 2.56 (3H, s, 7-Me), 2.62 (3H, s, 2-SMe),

4.94 (2H, q, J = 7.2 Hz, 10-CH₂-CH₃), 7.69–7.74 (1H,

m, 9-H), 7.76–7.81 (3H, m, 6, 8-H), 9.17 (1H, s, 5-H);

Anal. calcd for C₁₅H₁₅N₃OS: C, 63.13; H, 5.30; N,

14.73. Found: C, 63.37; H, 5.55; N, 14.51.

4.1.21.

7,8,10-Trimethyl-2-methylthiopyrimido[4,5-b]-

quinolin-4(10H)-one (2i). Yield, (2.31 g, 81%); mp 266–

268 °C (decomp. from DMF); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 226 (4.65), 270 sh (4.35), 284

(4.48), 351 (4.23), 430 (4.30); IR (m_max/cm^ꢀ1): 1655

4.1.26.

7-Methoxy-10-methyl-2-dimethylaminopyrimi-

do[4,5-b]quinolin-4(10H)-one (3c). Yield, (1.24 g, 87%);

mp 291–294 °C (decomp. from DMF); UV (EtOH):

1

(CO); H NMR (CDCl₃): d 2.46 (3H, s, 7-Me), 2.57

k

_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1): 225 (4.64), 279 (4.69),

(3H, s, 8-Me), 2.62 (3H, s, 2-SMe), 4.26 (3H, s, 10-

Me), 7.56 (1H, s, 9-H), 7.70 (1H, s, 6-H), 9.13 (1H, s,

5-H); Anal. calcd for C₁₅H₁₅N₃OS: C, 63.13; H, 5.30;

N, 14.72. Found: C, 62.83; H, 5.42; N, 14.32.

326 (4.18), 400sh (4.09), 428 (4.37), 454 (4.36); IR

(m_max/cm^ꢀ1): 1660 (CO); ¹H NMR (CDCl₃): d 3.33

(6H, s, 2-N(CH₃)₂), 3.92 (3H, s, 7-OMe), 4.12 (3H,

s,10-Me), 7.20 (1H, d, J_6,8= 3.0 Hz, 6-H), 7.44 (1H,

dd, J_8,9= 9.3 Hz, J _6,8= 3.0 Hz, 8-H), 7.60 (1 H, d,

J_8,9= 9.3 Hz, 9-H), 8.88 (1 H, s, 5-H); Anal. calcd for

C₁₅H₁₆N₄O₂: C, 63.37; H, 5.67; N, 19.71. Found: C,

63.03; H, 5.65; N, 19.52.

4.1.22. 6,9-Dimethoxy-10-methyl-2-methylthiopyrimido-

[4,5-b]quinolin-4(10H)-one (2j). Yield, (2.73 g, 86%); mp

251–253 °C (decomp. from DMF); UV (EtOH): k_max

/

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H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

4.1.27. 7-Chloro-10-methyl-2-dimethylaminopyrimido-

[4,5-b]quinolin-4(10H)-one (3d). Yield, (0.94 g, 65%); mp

m, 8 and 9-H), 8.20 (1H, dd, J _6,7= 7.8 Hz,

J_6,8= 1.2 Hz, 6-H), 8.92 (1 H, s, 5-H); Anal. calcd for

C₁₃H₁₂N₄OÆH₂O: C, 60.45; H, 5.46; N, 21.69. Found:

C, 60.28; H, 5.49; N, 21.29.

291–293 °C (decomp. from DMF); UV (EtOH): k_max

/

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 225 (4.64), 275 (4.70),

286sh (4.52), 318 (4.06), 390sh (4.10), 415 (4.25), 424

(4.19); IR (m_max/cm^ꢀ1): 1650 (CO); ¹H NMR (CDCl₃): d

3.33 (3H, s, 2-NMe), 3.34 (3H, s, 2-NMe), 4.09 (3H,

s,10-Me), 7.59 (1H, d, J_8,9= 9.0 Hz, 9-H), 7.74 (1H, dd,

J_8,9= 9.0 Hz, J_6,8= 2.4 Hz, 8-H), 7.80 (1H, d,

J_6,8= 2.4 Hz, 6-H), 8.81 (1H, s, 5-H); Anal. calcd for

C₁₄H₁₃ClN₄OÆ0.2H₂O: C, 57.52; H, 4.62; N, 19.17.

Found: C, 57.40; H, 4.60; N, 19.07.

4.1.32.

2-Amino-7,10-dimethylpyrimido[4,5-b]quinolin-

4(10H)-one (4c). Yield, (1.02 g, 85%); mp >300 °C (from

DMF); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

221 (4.57), 268 (4.66), 320 (4.08), 381sh (3.96), 408

(4.11), 428 (4.02); IR (m_max/cm^ꢀ1): 3360 and 3330

1

(NH), 1635 (CO); H NMR [(CD₃)₂SO]: d 2.47 (3H, s,

7-Me), 4.09 (3H, s, 10-Me), 7.15 (2H, br s, 2-NH₂,

exchangeable with D₂O), 7.75 (1H, dd, J_8,9= 9.0 Hz,

J_6,8= 2.1 Hz, 8-H), 7.82 (1H, d, J_8,9= 9.0 Hz, 9-H),

7.94 (1 H, br s, 6-H), 8.79 (1H, s, 5-H).; Anal. calcd

for C₁₃H₁₂N₄OÆ0.3H₂O: C, 63.56; H, 5.17; N, 22.81.

Found: C, 63.96; H, 5.34; N, 22.44.

4.1.28. 7,10-Dimethyl-2-dimethylaminopyrimido[4,5-b]-

quinolin-4(10H)-one (3e). Yield, (1.15 g, 86%); mp

>300 °C (decomp. from DMF); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 222 (4.68), 273 (4.68), 281sh

(4.59), 323 (4.13), 389sh (4.15), 413 (4.35), 438 (4.30); IR

1

(m_max/cm^ꢀ1): 1655 (CO); H NMR (CDCl₃): d 2.50 (3H,

4.1.33. General procedure for the preparation of 10-alkyl-

5-deazaﬂavins {10-alkylpyrimido[4,5-b]quinoline-2,4(3H,

10H)-diones} (5). To 10-alkyl-2-deoxo-2-methylthio-5-

deazaﬂavins (2, 0.01 mol) was added 5N hydrochloric

acid (250 mL). Then, the mixture was reﬂuxed for 1–2

h and the resulting clear yellow solution was concen-

trated in vacuo. The yellow residue was crushed with

water and neutralized with aqueous ammonia (pH 7).

The precipitated powdery crystals were ﬁltered oﬀ,

washed well with water, dried, and recrystallized from

glacial acetic acid to aﬀord the corresponding products

as yellow needles in 78–91% yields.

s, 7-Me), 3.33 (6H, s, 2-NMe₂), 4.10 (3H, s,10-Me), 7.55

(1H, d, J_8,9= 9.6 Hz, 9-H), 7.61–7.65 (2H, m, 6 and 8-

H), 8.88 (1H, s, 5-H); Anal. calcd for C₁₅H₁₆N₄O: C,

67.15; H, 6.01; N, 20.88. Found: C, 66.92; H, 6.09; N,

20.88.

4.1.29. General procedure for the preparation of 10-alkyl-

2-amino-2-deoxo-5-deazaﬂavins {10-alkyl-2-aminopyrim-

ido[4,5-b]quinolin-4(10H)-ones} (4a–c). A mixture of 10-

alkyl-2-deoxo-2-methylthio-5-deazaﬂavins (2, 4.0 mmol)

and ammonium acetate (10 g, 0.13 mol) was heated un-

der stirring at 160–165 °C for 0.5–3 h. The reaction mix-

ture was cooled, diluted with water (15 mL), neutralized

with aqueous ammonia (pH 7), and cooled in refrigera-

tor overnight. The resulting yellow crystals were col-

lected by ﬁltration, dried, and crystallized from an

appropriate solvent to give the corresponding products

as yellow needles in 57–77% yields.

4.1.34. 10-Methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-

dione (5a). Yield, (2.07 g, 91%); mp >300 °C (from

HOAc, lit.,²⁷359 °C); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 218 (4.57), 261 (4.55), 318 (3.97),

398 (4.04), 420 (3.88); IR (m_max/cm^ꢀ1): 3140 (NH),

1700 and 1660 (CO); ¹H NMR [(CD₃)₂SO]: d 4.04

(3H, s, 10-Me), 7.48–7.61 (1H, br m, 7-H), 7.86–8.03

(2H, br m, 8 and 9-H), 8.18 (1H, br d, J_6,7= 7.2 Hz,

6-H), 9.0 (1 H, s, 5-H), 11.06 (1H, br s, 3-NH, exchange-

able with D₂O); Anal. calcd for C₁₂H₉N₃O₂Æ0.2H₂O: C,

62.44; H, 4.10; N, 18.20. Found: C, 62.16; H, 4.24; N,

17.92.

4.1.30. 2-Amino-10-methylpyrimido[4,5-b]quinolin-4(10H)-

one (4a). Yield, (0.64 g, 57%); mp 284–288 °C (decomp.

from DMF, lit.,²⁶288 °C); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 218 (4.40), 267 (4.44), 319 (3.95),

380sh (3.67), 401 (3.83), 425 (3.72); IR (m_max/cm^ꢀ1): 3340

1

and 3470 (NH), 1640 (CO); H NMR (CDCl₃): d 4.18

(3H, s,10-NMe), 5.38 (1H, br s, 2-NH, exchangeable with

D₂O), 5.74 (1H, br s, 2-NH,exchangeable with D₂O), 7.43

(1H, dt, J_6,7= J_7,8= 7.8 Hz, J_7,9= 1.5 Hz, 7-H), 7.71 (1H,

d, J_8,9= 8.7 Hz, 9-H), 7.88 (1H, dt, J_7,8= 7.8 Hz,

J_8,9= 8.7 Hz, J_6,8= 1.5 Hz, 8-H), 7.90 (1 H, dd,

J_6,7= 7.8 Hz, J_6,8= 1.5 Hz, 6-H), 9.06 (1H, s, 5-H); Anal.

calcd for C₁₂H₁₀N₄OÆ0.3H₂O: C, 62.22; H, 4.61; N, 24.19.

Found: C, 62.35; H, 4.65; N, 23.78.

4.1.35. 10-Ethylpyrimido[4,5-b]quinoline-2,4(3H,10H)-

dione (5b). Yield, (2.20 g, 91%); mp >300 °C (from HOAc,

lit.,²⁷353 °C); UV (EtOH):

k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 219 (4.43), 262 (4.65), 319 (4.05), 396

(3.17), 417 (4.06); IR (m_max/cm^ꢀ1): 3140 (NH), 1700 and

1660 (CO); ¹H NMR [(CD₃)₂SO]: d 1.34 (3H, t,

J = 7.2 Hz, 10-CH₂-CH₃), 4.74 (2 H, q, J = 7.2 Hz, 10-

CH₂-CH₃),

7.54

(1H,

dt,

J_6,7= J_7,8= 8.1 Hz,

J_7,9= 2.4 Hz, 7-H), 7.92–8.01 (2H, m, 8 and 9-H), 8.19

(1H, br d, J_6,7= 8.1 Hz, 6-H), 9.0 (1H, s, 5-H), 11.08

(1H, br s, 3-NH, exchangeable with D₂O); Anal. calcd

for C₁₃H₁₁N₃O₂: C, 64.72; H, 4.60; N, 17.42. Found: C,

64.88; H, 4.85; N, 17.05.

4.1.31. 2-Amino-10-ethylpyrimido[4,5-b]quinolin-4(10H)-

one (4b). Yield, (0.93 g, 77%); mp 264–266 °C (from

EtOH, lit.,²⁶266 °C); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 220 (4.56), 268 (4.63), 319 (4.09),

378sh (3.83), 400 (4.02), 424 (3.91); IR (m_max/cm^ꢀ1):

1

3320 and 3410 (NH), 1630 (CO); H NMR [(CD₃)₂SO]:

4.1.36.

7-Methoxy-10-methylpyrimido[4,5-b]quinoline-

d 1.36 (3H, t, J = 6.9 Hz, 10-CH₂-CH₃), 4.81 (2H, q,

J = 6.9 Hz, 10-CH₂-CH₃), 7.38 (2H, br s, 2-

NH_2,exchangeable with D₂O), 7.54 (1 H, dt,

J_6,7= J_7,8= 7.8 Hz, J_7,9= 1.2 Hz, 7-H), 7.88–8.06 (2H,

2,4(3H,10H)-dione (5c). Yield, (2.01 g, 91%); mp

>300 °C (from HOAc); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 225 (4.69), 262 (4.77), 324 (4.39),

426 (4.41), 447 (4.40); IR (m_max/cm^ꢀ1): 3140 (NH),

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6349

1700 and 1655 (CO); ¹H NMR [(CD₃)₂SO]: d 3.91 (3H, s,

7-OMe), 4.05 (3H, s, 10-Me), 7.59 (1H, dd, J_6,8= 2.7 Hz,

J_8,9= 8.7 Hz, 8-H), 7.70 (1H, d, J_6,8= 2.7 Hz, 6-H), 7.87

(1H, d, J_8,9= 8.7 Hz, 9-H), 8.90 (1H, s, 5-H), 10.65 (1H,

br s, 3-NH, exchangeable with D₂O); Anal. calcd for

C₁₃H₁₁N₃O₃Æ0.1H₂O: C, 60.27; H, 4.36; N, 16.22. Found:

C, 60.25; H, 4.68; N, 15.83.

(4.18), 431 (4.12); IR (m_max/cm^ꢀ1): 3260 (OH), 1650

(CO); ¹H NMR (CDCl₃): d 2.01 (1H, br s, OH,

exchangeable with D₂O), 3.39 (3H, s, 2-NMe), 3.96

(4H, br s, 2-N-CH₂-CH₂), 4.12 (3H, s, 10-Me), 7.44

(1H, t, J_6,7= J_7,8= 8.1 Hz, 7-H), 7.65 (1H, d,

J_8,9= 8.1 Hz, 9-H), 7.82 (1H, t, J_7,8= J_8,9= 8.1 Hz, 8-

H), 7.89 (1H, d, J_6,7= 8.1 Hz, 6-H), 8.95 (1H, s, 5-H);

Anal. calcd for C₁₅H₁₆N₄O₂Æ0.4 H₂O: C, 61.80; H,

5.81; N, 19.22. Found: C, 61.93; H, 5.71; N, 18.91.

4.1.37. Preparation of 2-n-butylamino-10-methylpyrimi-

do[4,5-b]quinolin-4-(10H)-one (6). n-Butylammonium

acetate was prepared in situ by slowly adding glacial

acetic acid (5.7 mL, 0.1 mol) to n-butylamine (7.31 g,

0.1 mol) in an ice bath. 2-Deoxo-10-methyl-2-methyl-

thio-5-deazaﬂavin (2, 0.008 mol) was added to the n-

butylammonium acetate and the mixture was fused un-

der stirring at 150–165 °C for 1–3 h. After the reaction

was complete, water (100 mL) was added to the mixture

to give a turbid solution. The residue concentrated in va-

cuo was dissolved in hot water and brought to pH 8 with

sodium bicarbonate to give a yellow crystalline solid,

which was dried, and recrystallized from DMF to aﬀord

yellow needles.

4.1.40. 10-Ethyl-2-[N-(2-hydroxyethyl)-N-methylamino]-

pyrimido[4,5-b]quinolin-4-(10H)-one (7b). Yield, (1.19 g ,

80%); mp 250–252 °C (from DMF); UV (EtOH): k_max

/

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 221 (4.72), 271 (4.68),

279sh (4.63), 319 (4.12), 384sh (4.22), 408 (4.39), 432

(4.32); IR (m_max/cm^ꢀ1): 3260 (OH), 1650 (CO); ¹H NMR

(CDCl₃): d 1.51 (3H, t, J = 6.9 Hz, 10-CH₂-CH₃), 1.81

(1H, br s, OH, exchangeable with D₂O), 3.40 (3H, s, 2-

NMe), 3.93–3.99 (4H, m, 2-NCH₂-CH₂-), 4.80 (2H, q,

J = 6.9 Hz, 10-CH₂-CH₃), 7.47 (1H, t, J_6,7= J_7,8

=

7.8 Hz, 7-H), 7.68 (1H, d, J_8,9= 7.8 Hz, 9-H), 7.85 (1H,

dt, J_7,8= J_8,9= 7.8 Hz, J_6,8= 1.5 Hz, 8-H), 7.93 (1H, d,

J_6,7= 7.8 Hz, 6-H), 8.97 (1H, s, 5-H); Anal. calcd for

C₁₆H₁₈N₄O₂: C, 64.41; H, 6.08; N, 18.78. Found: C,

64.13; H, 6.06; N, 18.50.

Yield, (1.28 g, 91%); mp 252–254 °C (decomp. from

DMF); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

219 (4.59), 269 (4.63), 273sh (4.60), 321 (4.08), 403

(4.13), 422sh (4.07); IR (m_max/cm^ꢀ1): 3180 (NH), 1655

4.1.41. 10-Ethyl-2-[N-(2-hydroxyethyl)-N-methylamino]-

7-methylpyrimido[4,5-b]quinolin-4-(10H)-one (7c). Yield,

(1.16 g, 74%); mp 272–274 °C (decomp. from DMF);

UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1): 224

(4.64), 274 (4.62), 282sh (4.53), 325 (4.09), 387sh

(4.09), 414 (4.30), 438 (4.26); IR (m_max/cm^ꢀ1): 3210

(OH), 1650 (CO); ¹H NMR (CDCl₃): d 1.31 (3H, t,

J = 6.9 Hz, 10-CH₂–CH₃), 1.81 (1H, br s, OH,

exchangeable with D₂O), 2.51 (3H, s, 7-Me), 3.39 (3H,

s, 2-NMe), 3.96 (4H, br s, 2-NCH₂–CH₂-), 4.77 (2H,

q, J = 6.9 Hz, 10-CH₂–CH₃), 7.54–7.61 (2H, m, 6 and

8-H), 7.66 (1H, d, J_8,9= 8.1 Hz, 9-H), 8.91 (1H, s, 5-

H); Anal. calcd for C₁₇H₂₀N₄O₂: C, 65.37; H, 6.45; N,

17.94. Found: C, 65.21; H, 6.46; N, 17.84.

1

(CO); H NMR [(CD₃)₂SO]: at 100 °C: d 0.93 (3 H, t,

J = 7.2 Hz, 2-N(CH₂)₃–CH₃), 1.39 (2H, sextet,

J = 7.2 Hz, 2-N(CH₂)₂–CH₂–CH₃), 1.59 (2H, quintet,

J = 7.2 Hz, 2-NCH₂–CH₂–CH₂–CH₃), 3.30–3.55 (2H,

m, 2-NCH₂–(CH₂)₂–CH₃), 4.07 (3H, s, 10-Me), 7.14

(1H, br s, 2-NH_,exchangeable with D₂O), 7.45–7.55

(1H, m, 9-H), 7.78–7.87 (2H, m, 7 and 8-H), 8.06 (1H,

br d, J = 7.8 Hz, 6-H), 8.77 (1H, s, 5-H); Anal. calcd

for C₁₆H₁₈N₄OÆ0.4H₂O: C, 66.37; H, 6.54; N, 19.35.

Found: C, 66.04; H, 6.52; N, 19.17.

4.1.38. General procedure for the preparation of 2-(substi-

tuted amino)-10-alkyl-2-deoxo-5-deazaﬂavins {2-(substi-

tuted

amino)-10-alkylpyrimido[4,5-b]quinolin-4(10H)-

ones} (7a–q). A mixture of 10-alkyl-2-deoxo-2-methyl-

thio-5-deazaﬂavins (2, 5.0 mmol) and an appropriate pri-

mary or secondary amine (25–35 mmol) in n-butanol

(30 mL) was reﬂuxed with stirring for 2–5 h. After the

clear yellow solution was kept overnight in refrigerator,

the precipitated yellow crystalline solid was collected by

ﬁltration to get the ﬁrst crop. The ﬁltrate was concen-

trated in vacuo and the residue was treated with appropri-

ate solvent, such as water or ethanol for the piperidine,

morpholine, N-(2-hydroxyethyl)-N-methylamine, and

N-methylpiperazine derivatives and petroleum ether for

the n-octylamine derivative, to get the second crop of

the crude product free from amines. The collected solids

were dried and recrystallized from an appropriate solvent

to give the corresponding products as bright yellow nee-

dles in 70–99% yields.

4.1.42. 2-[N-(2-Hydroxyethyl)-N-methylamino]-8-meth-

oxy-10-methylpyrimido[4,5-b]quinolin-4(10H)-one (7d).

Yield, (1.19 g, 76%); mp 288–291 °C (decomp. from

DMF); UV (EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1):

230 (4.69), 269 (4.53), 274sh (4.50), 354sh (4.04), 380sh

(4.18), 401 (4.46), 423 (4.50); IR (m_max/cm^ꢀ1): 3200

1

(OH), 1645 (CO); H NMR (CDCl₃): d 3.31 (3H, s, 2-

NMe), 3.58–3.74 (2H, m, 2-N-CH₂-CH₂), 3.75–3.84

(2H, m, 2-N-CH₂-CH₂), 4.00 (3H, s, 8-OMe), 4.09

(3H, s,10-Me), 4.64 (1H, br s, OH, exchangeable with

D₂O), 7.15 (1H, br d, J_6,7= 8.4 Hz, 7-H), 7.24 (1 H,

br s, 9-H), 8.06 (1H, d, J_6,7= 8.4 Hz, 6-H), 8.78 (1H,

s, 5-H); Anal. calcd for C₁₆H₁₈N₄O₃.0.1H₂O: C, 60.79;

H, 5.80; N, 17.72. Found: C, 60.72; H, 5.77; N, 17.65.

4.1.43. 10-Methyl-2-piperidinopyrimido[4,5-b]quinolin-

4(10H)-one (7e). Yield, (1.19 g, 81%); mp 275–277 °C

4.1.39. 2-[N-(2-Hydroxyethyl)-N-methylamino]-10-meth-

ylpyrimido[4,5-b]quinolin-4(10H)-one (7a). Yield, (1.11 g,

78%); mp 251–253 °C (decomp. from DMF); UV

(EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1): 221 (4.51),

272 (4.49), 278sh (4.45), 321 (3.92), 383sh (4.00), 408

(from DMF); UV (EtOH):

dm³mol^ꢀ1cm^ꢀ1): 221 (4.62), 273 (4.62), 278sh (4.60),

k_max/nm (log e/

320 (4.03), 384sh (4.15), 410 (4.31), 434 (4.24); IR (m_max

/

1

cm^ꢀ1): 1660 (CO); H NMR (CDCl₃): d 1.68 (6H, br s,

3⁰, 4⁰and 5⁰-CH₂), 3.98–4.07 (4H, m, 2⁰and 6⁰-CH₂),

6350

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

4.11 (3H, s, 10-Me), 7.44 (1H, dt, J_6,7= 8.7 Hz,

J_7,8= 7.8 Hz, J_7,9= 1.5 Hz, 7-H), 7.64 (1 H, d,

J_8,9= 8.7 Hz, 9-H), 7.80 (1H, dt, J_7,8= 7.8, Hz

J_8,9= 8.7 Hz, J_6,8= 1.5 Hz, 8-H), 7.87 (1H, dd,

J_6,7= 8.7 Hz, J_6,8= 1.5 Hz, 6-H), 8.93 (1H, s, 5-H); Anal.

calcd for C₁₇H₁₈N₄OÆ0.1H₂O: C, 68.94; H, 6.19; N, 18.92.

Found: C, 68.66; H, 6.17; N, 18.89.

4.1.48. 7,10-Dimethyl-2-piperidinopyrimido[4,5-b]quino-

lin-4-(10H)-one (7j). Yield, (1.48 g, 96%); mp 292–

293 °C (from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 222 (4.72), 275 (4.71), 283sh (4.64),

322 (4.13), 391sh (4.18), 415 (4.38), 440 (4.33); IR

(m_max/cm^ꢀ1): 1640 (CO); ¹H NMR (CDCl₃): d 1.67

(6H, br s, 3⁰, 4⁰and 5⁰-CH₂), 2.49 (3H, s, 7-Me), 3.97–

4.06 (4H, m, 2⁰and 6⁰-CH₂), 4.09 (3H, s, 10-Me), 7.53

(1H, d, J_8,9= 9.3 Hz, 9-H), 7.58–7.65 (2H, m, 6 and 8-

H), 8.86 (1H, s, 5-H); Anal. calcd for C₁₈H₂₀N₄OÆ1.1-

H₂O: C, 65.87; H, 6.82; N, 17.07. Found: C, 66.16; H,

7.22; N, 17.16.

4.1.44.

7-Chloro-10-methyl-2-morpholinopyrimido[4,5-

b]quinolin-4(10H)-one (7f). Yield, (1.29 g, 78%), mp

>300 °C (from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 225 (4.65), 276 (4.61), 286sh (4.53),

319 (4.02), 392sh (4.07), 416 (4.23), 441 (4.19); IR (m_max

/

cm^ꢀ1): 1660 (CO); H NMR (CDCl₃): d 3.73–3.82 (4H,

m, 2⁰and 6⁰-NCH₂), 4.03-4.15 (4H, m, 3⁰and 5⁰-OCH₂),

4.11 (3H, s, 10-Me), 7.61 (1H, d, J_8,9= 9.0 Hz, 9-H),

7.77 (1H, dd, J_8,9= 9.0 Hz, J_6,8= 2.4 Hz, 8-H), 7.85

(1H, d, J_6,8= 2.4 Hz, 6-H), 8.87 (1H, s, 5-H); Anal. calcd

for C₁₆H₁₅ClN₄O₂Æ0.4H₂O: C, 56.86; H, 4.71; N, 16.58.

Found: C, 56.86; H, 4.61; N, 16.24.

4.1.49. 10-Ethyl-7-methyl-2-morpholinopyrimido[4,5-b]-

quinolin-4-(10H)-one (7k). Yield, (1.27 g, 78%); mp

>300 °C (from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 225 (4.63), 274 (4.62), 283sh (4.55),

326 (4.12), 387sh (4.09), 413 (4.32), 437 (4.29); IR

(m_max/cm^ꢀ1): 1650 (CO); ¹H NMR (CDCl₃): d 1.48

(3H, t, J = 7.2 Hz, 10-CH₂-CH₃), 2.51 (3H, s, 7-Me),

3.70–3.85 (4H, m, 2⁰and 6⁰-NCH₂), 3.99–4.16 (4 H,

m, 3⁰and 5⁰-OCH₂), 4.76 (2H, q, J = 7.2 Hz, 10-CH₂-

CH₃), 7.48 (1H, d, J_8,9= 9.3 Hz, 9-H), 7.64–7.68 (2H,

m, 6 and 8-H), 8.93 (1H, s, 5-H); Anal. calcd for

C₁₈H₂₀N₄O₂: C, 66.65; H, 6.21; N, 17.27. Found: C,

66.44; H, 6.37; N, 17.40.

1

4.1.45. 10-Methyl-2-morpholinopyrimido[4,5-b]quinolin-

4-(10H)-one (7g). Yield, (1.47 g, 99%); mp 296–298 °C

(decomp. from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 220 (4.63), 272 (4.60), 279sh (4.55),

319 (4.07), 383sh (4.08), 406 (4.26), 431 (4.20); IR (m_max

/

1

cm^ꢀ1): 1650 (CO); H NMR (CDCl₃): d 3.74–3.83 (4H,

m, 2⁰and 6⁰-NCH₂), 4.04–4.13 (4H, m, 3⁰and 5⁰-

4.1.50.

7-Chloro-10-methyl-2-(4-methylpiperazinyl)-

OCH₂), 4.14 (3H, s,10-Me), 7.48 (1H, t, J_6,7

=

pyrimido[4,5-b]quinolin-4-(10H)-one (7l). Yield, (1.46 g,

85%); mp >300 °C (decomp. from DMF); UV (EtOH):

J_7,8= 7.8 Hz, 7-H), 7.68 (1H, d, J_8,9= 8.7 Hz, 9-H), 7.85

(1H, dt, J_7,8= 7.8 Hz, J_8,9= 8.7 Hz, J_6,8= 1.5 Hz, 8-H),

7.91 (1 H, d, J_6,7= 7.8 Hz, 6-H), 8.99 (1 H, s, 5-H); Anal.

calcd for C₁₆H₁₆N₄O₂Æ0.4 H₂O: C, 63.31; H, 5.58; N,

18.46. Found: C, 63.61; H, 5.84; N, 18.41.

k

_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1): 225 (4.65), 275 (4.65),

284sh (4.59), 319 (4.02), 390sh (4.09), 416 (4.27), 442

1

(4.21); IR (m_max/cm^ꢀ1): 1660 (CO); H NMR (CDCl₃):

d 2.34 (3H, s, 4⁰-Me), 2.45–2.53 (4H, m, 3⁰and 5⁰-

CH₂), 4.05–4.15 (4H, m, 2⁰and 6⁰-CH₂), 4.10 (3H, s,

10-Me), 7.60 (1H, d, J_8,9= 9.0 Hz, 9-H), 7.75 (1H, dd,

J_8,9= 9.0 Hz, J_6,8= 2.1 Hz, 8-H), 7.81 (1 H, d,

J_6,8= 2.1 Hz, 6-H), 8.82 (1H, s, 5-H); Anal. calcd for

C₁₇H₁₈ClN₅OÆ0.1H₂O: C, 59.08; H, 5.31; N, 20.26.

Found: C, 58.72; H, 5.43; N, 20.69.

4.1.46. 10-Methyl-2-(4-methylpiperazinyl)pyrimido[4,5-

b]quinolin-4-(10H)-one (7h). Yield, (1.08 g, 70%); mp

235–237 °C (decomp. from DMF); UV (EtOH): k_max

/

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 220 (4.56), 272 (4.56),

277sh (4.53), 322 (3.99), 382sh (4.06), 407 (4.24), 430

(4.17); IR (m_max/cm^ꢀ1): 1650 (CO); ¹H NMR (CDCl₃): d

2.35 (3H, br s, 4⁰-Me), 2.42–2.58 (4H, m, 3⁰and 5⁰-

CH₂), 4.06–4.11 (4H, m, 2⁰and 6⁰-CH₂), 4.13 (3H, s,10-

Me), 7.46 (1H, t, J_6,7= J_7,8= 7.8 Hz, 7-H), 7.68 (1H, d,

J_8,9= 9.0 Hz, 9-H), 7.83 (1H, dt, J_7,8= 7.8 Hz,

J_8,9= 9.0 Hz, J_6,8= 1.5 Hz, 8-H), 7.91 (1H, d,

J_6,7= 7.8 Hz, 6-H), 8.96 (1H, s, 5-H); Anal. calcd for

C₁₇H₁₉N₅OÆ0.4H₂O: C, 64.50; H, 6.30; N, 22.12. Found:

C, 64.28; H, 6.22; N, 21.92.

4.1.51. 7-Chloro-10-methyl-2-piperidinopyrimido[4,5-b]-

quinolin-4-(10H)-one (7m). Yield, (1.46 g, 89%); mp

>300 °C (from DMF); UV (EtOH): k_max/nm (log e/

dm³mol^ꢀ1cm^ꢀ1): 226 (4.77), 276 (4.78), 286sh (4.71),

317 (4.11), 393sh (4.24), 420 (4.41), 445 (4.35); IR

(m_max/cm^ꢀ1): 1645 (CO); ¹H NMR (CDCl₃): d 1.70

(6H, br s, 3⁰, 4⁰and 5⁰-CH₂), 3.96–4.06 (4H, m, 2⁰and

6⁰-NCH₂), 4.08 (3H, s, 10-Me), 7.57 (1H, d,

J_8,9= 9.0 Hz, 9-H), 7.72 (1H, d, J_8,9= 9.0 Hz, 8-H),

7.79 (1H, s, 6-H), 8.79 (1H, s, 5-H); Anal. calcd for

C₁₇H₁₇ClN₄OÆ1.1H₂O: C, 58.57; H, 5.55; N, 16.07.

Found: C, 58.23; H, 5.46; N, 15.82.

4.1.47. 7-Methoxy-10-methyl-2-morpholinopyrimido[4,5-

b]quinolin-4-(10H)-one (7i). Yield, (1.57 g, 96%); mp

296–299 °C (decomp. from DMF); UV (EtOH): k_max

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 226 (4.47), 276 (4.49), 328

(4.03), 400sh (3.92), 427 (4.16), 453 (4.18); IR (m_max

/

4.1.52.

quinolin-4-(10H)-one (7n). Yield, (1.28 g, 79%); mp

287–289 °C (decomp. from DMF); UV (EtOH): k_max

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 225 (4.75), 276 (4.70), 336

7,8,10-Trimethyl-2-morpholinopyrimido[4,5-b]-

cm^ꢀ1): 1655 (CO); ¹H NMR (CDCl₃): d 3.72–3.82

(4H, m, 2⁰and 6⁰-NCH₂), 3.93 (3H, s, 7-OMe), 4.00–

4.12 (4H, m, 3⁰and 5⁰-OCH₂), 4.13 (3H, s, 10-Me),

7.22 (1H, d, J_6,8= 2.7 Hz, 6-H), 7.47 (1H, dd,

J_8,9= 9.3 Hz, J_6,8= 2.7 Hz, 8-H), 7.68 (1H, d,

J_8,9= 9.3 Hz, 9-H), 8.91 (1H, s, 5-H); Anal. calcd for

C₁₇H₁₈N₄O₃ÆH₂O: C, 59.29; H, 5.85; N, 16.27. Found:

C, 59.30; H, 5.76; N, 15.89.

/

(4.18), 387sh (4.15), 410 (4.38), 434 (4.67); IR (m_max

/

1

cm^ꢀ1): 1650 (CO); H NMR (CDCl₃): d 2.41 (3H, s, 7-

Me), 2.52 (3H, s, 8-Me), 3.73–3.81 (4H, m, 2⁰and 6⁰-

NCH₂), 4.02–4.09 (4H, m, 3⁰and 5⁰-OCH₂), 4.11 (3H,

s, 10-Me), 7.44 (1H, s, 9-H), 7.60 (1H, s, 6 H), 8.91

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

6351

(1H, s, 5-H); Anal. calcd for C₁₈H₂₀N₄O₂Æ0.4H₂O: C,

65.20; H, 6.32; N, 16.90. Found: C, 65.05; H, 6.38; N,

17.10.

4.3. Molecular docking study

The advanced docking program AutoDock 3.0.5²⁴was

used to evaluate the binding free energy of the inhibitors

within the macromolecules. The individual components

of the program include AutoTors, AutoGrid, and Auto-

Dock. AutoTors deﬁnes which bonds in the ligand are

rotatable, aﬀecting the degrees of freedom (DOF) of

the ligand and hence the complexity of computations.

AutoGrid pre-calculates a three-dimensional grid of

interaction energies based on the macromolecular target

using the AMBER force ﬁeld. AutoDock performs the

task of the docking. First, the ligand moves randomly

in any one of six degrees of freedom, namely 3 transla-

tion degrees and 3 rotation degrees, and the energy of

the new ligand ‘state’ is calculated. If the energy of the

new state is lower than that of the old state, the new

one is automatically accepted as the next step in

docking.

4.1.53.

7-Methoxy-10-methyl-2-(4-methylpiperazinyl)-

pyrimido[4,5-b]quinolin-4-(10H)-one (7o). Yield, (1.54 g,

85%); mp 237–239 °C (decomp. from DMF); UV

(EtOH): k_max/nm (log e/dm³mol^ꢀ1cm^ꢀ1): 226 (4.60),

280 (4.68), 327 (4.18), 400sh (4.05), 427 (4.32), 453

1

(4.34); IR (m_max/cm^ꢀ1): 1640 (CO); H NMR (CDCl₃):

d 2.34 (3 H, br s, 4⁰-Me), 2.45–2.53 (4H, m, 3⁰and 5⁰-

CH₂), 3.92 (3H, s, 7-OMe), 4.04–4.10 (4H, m, 2⁰and

6⁰-CH₂), 4.12 (3H, s, 10-Me), 7.20 (1H, d,

J_6,8= 2.7 Hz, 6-H), 7.45 (1H, dd, J_8,9= 9.6 Hz,

J_6,8= 2.7 Hz, 8-H), 7.61 (1H, d, J_8,9= 9.6 Hz, 9-H),

8.89 (1H, s, 5-H); Anal. calcd for C₁₈H₂₁N₅O₂Æ0.4H₂O:

C, 62.38; H, 6.34; N, 20.21. Found: C, 62.65; H, 6.23;

N, 19.80.

4.1.54. 10-Ethyl-2-(2-hydroxyethylamino)pyrimido[4,5-b]-

quinolin-4-(10H)-one (7p). Yield, (1.14 g, 80%); mp

4.3.1. Preparation of ligands and target protein tyrosine

kinase. The compounds involved in this study as ligands

267–270 °C (decomp. from DMF); UV (EtOH): k_max

/

nm (log e/dm³mol^ꢀ1cm^ꢀ1): 221 (4.42), 268 (4.44),

274sh (4.41), 321 (3.90), 404sh (4.01), 425sh (3.93); IR

(m_max/cm^ꢀ1): 3250 (OH), 3190 (NH), 1640 (CO);¹H

NMR [(CD₃)₂SO] at 90 °C: d 1.40 (3H, t, J = 6.9 Hz,

10-CH₂-CH₃), 3.39–3.50 (2H, m, 2-NH-CH₂-CH₂-

OH), 3.56–3.62 (2H, m, 2-NH-CH₂-CH₂-OH), 3.96

(1H, br s, 2-NH-CH₂-CH₂-OH, exchangeable with

D₂O), 4.80 (2H, q, J = 6.9 Hz, 10-CH₂-CH₃), 7.19 (1H,

br s, 2-NH,exchangeable with D₂O), 7.44–7.58 (1H, m,

9-H), 7.84–7.98 (1H, m, 7 and 8-H), 8.15 (1H, br d,

J_6,7= 8.1 Hz, 6-H), 8.80 (1H, s, 5-H); Anal. calcd for

C₁₅H₁₆N₄O₂: C, 63.37; H, 5.67; N, 19.71. Found: C,

63.19; H, 5.73; N, 19.48.

include

10-alkyl-2-deoxo-2-methylthio-5-deazaﬂavins

(2a–j), 2-amino- and 2-(N-substituted amino)-10-alkyl-

2-deoxo-5-deazaﬂavins (3,4,6, and 7), and the computa-

tionally designed compounds connected with amino

acids (8a,b) were studied for their binding activities into

PTK. Since ligands are not peptides, Gasteiger-Huckel

¨

charge was assigned and then non-polar hydrogens were

merged. The rigid roots of each ligands were deﬁned

automatically instead of picking manually. The amide

bonds were made non-rotatable. The three-dimensional

structures of the aforementioned compounds were con-

structed using Chem3D ultra 8.0 software [Chemical

Structure Drawing Standard; Cambridge Soft corpora-

tion, USA (2003)], then they were energetically mini-

mized by using MOPAC with 100 iterations and

minimum RMS gradient of 0.10. The crystal structures

of c-Kit receptor protein-tyrosine kinase in complex

with STI-571 (Imatinib or Gleevec) were extracted from

the RCSB Protein Data Bank http://www.rcsb.org/pdb/

Welcome.do ccessed on August, 20, 2005. All bound

waters, ligand were removed from the protein. For the

target, the amino acids of the ligand binding site were

deﬁned using data in pdbsum http://www.ebi.ac.uk/

thornton-srv/databases/pdbsum/ accessed on January

8, 2006. The polar hydrogen model was chosen for the

crystal structure. The proteins were assigned with Koll-

man-united charge. The polar hydrogens and charges

were added.

4.1.55. 10-Methyl-2-n-octylaminopyrimido[4,5-b]quino-

lin-4-(10H)-one (7q). Yield, (1.46 g, 86%); mp 183–

185 °C (decompo, from EtOH); UV (EtOH): k_max/nm

(log e/dm³mol^ꢀ1cm^ꢀ1): 220 (4.60), 269 (4.63), 274sh

(4.60), 320 (4.07), 404 (4.19), 422sh (4.15); IR (m_max

/

cm^ꢀ1): 3180 (NH), 1655 (CO); H NMR [(CD₃)₂SO] at

90 °C: d 0.86 (3H, t, J = 7.2 Hz, 2-N(CH₂)₇-CH₃),

1.24–1.42 (10H, m, 2-N(CH₂)₂ꢀ (CH₂)₅-CH₃), 1.65

(2H, m, 2-NCH₂-CH₂-(CH₂)₅-CH₃), 3.40–3.61 (2H, m,

2-NCH₂-(CH₂)₆-CH₃), 4.21 (3H, s, 10-Me), 7.52–7.74

(1H, m, 7-H), 7.90–8.12 (2H, m, 8 and 9-H), 8.14–8.30

(1H, m, 6-H), 8.85 (1H, br s, 2-NH_,exchangeable with

D₂O), 9.12 (1H, s, 5-H); Anal. calcd for C₂₀H₂₆N₄O:

C, 70.98; H, 7.74; N, 16.55. Found: C, 70.66; H, 7.59;

N, 16.26.

1

4.3.2. Grid generation and run of molecular docking. The

grid maps representing the native ligand in the actual

docking target site were calculated with AutoGrid.

The grids (one for each atom type in the ligand, plus

one for electrostatic interactions) were chosen to be suf-

ﬁciently large to include not only the active site but also

signiﬁcant portions of the surrounding surface. The

three-dimensional grids, 80 A grid size (x, y, z) with a

˚

spacing of 0.375 A, were created because the location

of the native ligand in the crystal structure was known.

The cubic grid box was centered in the catalytic active

region and encompassed the binding site where the

4.2. Growth inhibitory activities of 2-substituted 10-alkyl-

2-deoxo-5-deazaﬂavins (2–4, 6, 7) and 5-deazaﬂavin (5)

against human tumor cell lines

The procedure was carried out using the modiﬁed MTT

assay²⁸to determine the inhibitory eﬀects of test com-

pounds on cell growth in vitro as mentioned in detail

in our previous paper.¹Two human tumor cell lines of

human T-cell acute lymphoblastoid leukemia (CCRF-

HSB-2) and human oral epidermoid carcinoma (KB)

were used in this study.

˚

6352

H. I. Ali et al. / Bioorg. Med. Chem. 15 (2007) 6336–6352

ligands were embedded. Then automated docking stud-

ies were carried out using AutoDock version 3.0.5.²⁴Of

the three diﬀerent search algorithms oﬀered by Auto-

Dock, the GA-LS search algorithm (Genetic algorithm

with local search) was chosen to search for the best con-

formers. The parameters were set using the software

ADT (Autodock Tool Kit) on PC which is associated

with Autodock 3.0.5.²⁴For all docking parameters, de-

fault values were used with 10 independent docking runs

for each docking case.

References and notes

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4.3.3. Molecular modeling and analysis of the docked

results. There are two kinds of free energies output by

Autodock. One is the predicted binding free energy that

includes the intermolecular energy and torsional free en-

ergy, and the other the docking energy that includes the

intermolecular and intramolecular energies. The former

is only reported at the end of a docking operation, while

the latter is used for selecting better individuals of pop-

ulation during a docking run.²⁴We used only the bind-

ing free energy of the ﬁrst type as the criterion for

ranking. Furthermore, the intermolecular hydrogen

bonds, whose eﬀect has already been counted in the

binding energy, were also investigated in order to ﬁnd

useful information for drug design. A comparison of

the results suggests that the binding free energy is more

reliable as a criterion for the virtual screening via molec-

ular docking. Cluster analysis was performed on the

docked results using a root mean square (RMS) toler-

ance of 0.5. Each of the clusters that exhibited signiﬁ-

cant negative interaction energies was examined by

Accelrys, DS modeling 1.7 [Accelrys Inc., San Diego,

CA (2006)] to determine their binding orientations,

molecular modeling, evaluation of the hydrogen bonds,

and for measuring RMSD, which was measured as dis-

tance between the centroids of the docked inhibitor and

the native ligand. The mode of interaction of the native

ligand within PTK was used as a standard docked model

as well as for RMSD calculation. The correct hydrogen

bond interaction was considered according to Taylor

et al.,²⁹who showed that C–H^{ꢁ ꢁ ꢁ}O in crystals contacts

14. Israel, M.; Protopapa, H. K.; Schlein, H. N.; Modest, E. J.

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˚

occur within certain distance (3.0–4.0 A) and angle (C–

H^{ꢁ ꢁ ꢁ}O, 90–180°) ranges. However, the more linear

hydrogen bond is likely to be more stronger.³⁰More-

over, there is general agreement that for carbonyl accep-

tors, the H^{ꢁ ꢁ ꢁ}O@C angle is distributed around 120°.

Therefore, in our modeling results we consider the

hydrogen bond angle P120° to be of a reasonable

strength.

23. Ali, H.I. Ph.D. thesis, Okayama University, 2007;

Okayama, Japan.

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Acknowledgment

The authors are indebted to the SC-NMR Laboratory

of Okayama University for the NMR spectral measure-

ments and the Okayama University Information

Technology Center for using Accelrys Discovery Studio

1.7.

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Article Doi

Antitumor studies. Part 3: Design, synthesis, antitumor activity, and molecular docking study of novel 2-methylthio-, 2-amino-, and 2-(N-substituted amino)-10-alkyl-2-deoxo-5-deazaflavins

DOI: 10.1016/j.bmc.2007.06.058

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Article abstract of DOI:10.1016/j.bmc.2007.06.058

Full text of DOI:10.1016/j.bmc.2007.06.058

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