Synthesis and biological investigation of pyrimido,1,2 a ,1,3,5,triazine and its analogues 1

The review is collectively categorized according to the common strategies in the synthesis of pyrimido[1,2-a][1,3,5]triazines: A annulation of pyrimidine onto a 1,3,5-triazine scaffold;

Chapter 1 INTRODUCTION

Nitrogen containing heterocycles are of immense importance biologically and industrially1 The triazines are among the oldest known organic nitrogen-containing heterocycles that possess three ‘N’ atoms in a six membered ring They may exist in three possible isomeric forms namely 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine (Fig 1)

1,2,3 -triazine

1,3,5 -triazine

1,2,4 -triazine

Fig 1 Isomeric forms of triazine (C3H3N3)

In the literature, there are many anticancer agents that possess the 1,3,5-triazine heterocycle Many of them are being used in the clinic and some are undergoing clinical trials (Fig 2) HMM (Hexamethylmelamine) is a substituted 1,3,5-triazine derivative which is used clinically as an alkylating anti-neoplastic agent for the treatment of ovarian cancer2 A190 is a dihydrotriazine derivative with anti-tumor activity against lung cancer The mechanism of A190 is known to be associated with blocking the G1 phase of the cell cycle and death by apoptosis3 ZSTK 474, a morpholinyl analogue, inhibits an ATP-competitive pan-class I phosphatidylinositol 3 kinase which plays a fundamental role in cellular responses such as proliferation, survival, motility and metabolism It has demonstrated antitumor activity against human cancer xenografts without showing toxic effects in critical organs4,5 Irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-1,3,5-triazine], an anti-gastric ulcer agent that is commonly used in Japan, has been shown to possess anti-angiogenic properties which contribute to the anti-cancer effect of the drug6 Dioxadet, a cytostatic agent, is used for the treatment of primary hepatic tumors and multiple intrahepatic metastases of colorectal carcinoma7 5-Aza-2'-deoxycytidine (decitabine)

is a DNA methyltransferase -I and -3B inhibitor It also stops silencing of the apoptotic BIK8

N N

N MeMe

N

Me

Me

N Me Me

Hexamethylmelamine

N N

N N

N N N

NH2

O

N N N

activity Some of these compounds include derivatives of pyrazolo[1,5-a]

[1,3,5]triazine (e.g 1 with IC50 (HCT 116) value = 0.99µM)9, 1,2,4-triazolo[1,5-a]

[1,3,5]triazine (e.g 2 with IC50 (MDA-MB-231) value = 28μM)10,11 and

1,3,5-triazino[1,2-a]benzimidazole12 (e.g 3 with IC50(DHFR) value =10.9μM) (Fig 3)

In the following section, a comprehensive literature review of the approaches for the

synthesis of pyrimido[1,2-a][1,3,5]triazines is presented It should be noted that the

latest review of this scaffold presented by Mahajan and coworkers13 in 2008 contained only 16 references and have missed out many valuable references

1.1 Literature review on the synthesis of pyrimido[1,2-a][1,3,5]-triazines and its

fused analogues

Ziegler and Noelken14 were the first to synthesize the pyrimido[1,2-a][1,3,5]triazine

system in 1961 (vide infra Scheme 1) The aim of this review is to summarize the

methods developed for the preparation of compounds with the

pyrimido[1,2-a][1,3,5]triazine scaffold (Fig 4) and polyfused system bearing this heterocyclic core

Information on the biological activity, if any, of pyrimido[1,2-a][1,3,5]triazine

derivatives is also included

N N N

1

3

4 5 6 7 8 9

Fig 4 Pyrimido[1,2-a][1,3,5]triazine scaffold

The review is collectively categorized according to the common strategies in the

synthesis of pyrimido[1,2-a][1,3,5]triazines: (A) annulation of pyrimidine onto a

1,3,5-triazine scaffold; (B) annulation of the 1,3,5-triazine ring onto a pyrimidine scaffold; (C) concurrent formation of both the 1,3,5-triazine and pyrimidine ring The

information on the biological activity of pyrimido[1,2-a][1,3,5]triazine derivatives is

also included

1.1.1 Synthesis by annulation of pyrimidine ring onto a 1,3,5-triazine scaffold

This synthetic strategy can be further classified into three methods as described below

Annulation of pyrimidine to amino[1,3,5]triazine with three carbon synthons

The reaction of 2-amino-1,3,5-triazines 4 with synthons (such as the malonates 5) that contribute three atoms to pyrimidine annulation would yield pyrimido[1,2-

a][1,3,5]triazines The regioselectivity of the ring closure in the formation of the

product was reported to be “questionable” by Ziegler and Noelken14 as equal

probability existed for the formation of 6 or 6’ However, the spectral support could

not be obtained Similarly, various 2,4-substituted

pyrimido[1,2-a][1,3,5]triazin-6,8-diones were synthesized and patented as insecticidal agents15 There is a limitation

using these malonates as only 6,8-dioxopyrimido[1,2-a][1,3,5]triazines can be

synthesized Much later, Katritzky and Yousaf16 studied the mechanism of dicarbonyl compounds with bis nucleophiles and concluded that the ring closure was faster than the initial nucleophilic attack without the isolation of the intermediate

1,3-(Scheme 1)

R1 = NH2, R2 = morpholinyl, R4 = H (6a); R1 = NH2, R2 = NMe2, R4 = H (6b); R1 = NH2, R2

= NEt2, R4 = H (6c); R1 = NH2, R2 = NH2, R4 = H (6d); R1 = NMe2, R2 = NMe2, R4 = H (6e)

Scheme 1: Reaction of 2-amino-1,3,5-triazines with malonates to give 2,4,7

substituted pyrimido[1,2-a][1,3,5]triazines (6a-f)

On the other hand, 1,4-dipolar cycloaddition of substituted

bis-(2,4,6-trichlorophenyl)malonates 5d17 to N-alkyl substituted amino 1,3,5-triazines 7 resulted

in the formation of mesoionic 6-oxo pyrimido[1,2-a]-1,3,5-triazin-9-ium-8-olates 8 in

high yields (Scheme 2) According to IUPAC Compendium of Chemical Terminology, mesoionic compounds are defined as “dipolar five or six membered heterocyclic compounds in which both the negative and the positive charge are delocalized and which cannot be represented satisfactorily by any one polar structure”18

Cyclocondensation of pyrimidine ring to give 6-oxo-7-carboxylic acid ester derivative

11 of pyrimido[1,2-a][1,3,5]triazine scaffold can be easily done using

ethoxymethylene malonate 10 as triatomic synthon Al-Shaar and co-workers19synthesized this fused tricyclic 1,3,5-triazine derivative 11 by adding imidazo[1,5-

a][1,3,5]triazin-4-amine to boiling ethoxymethylene malonate (Scheme 3)

O OEt

OEt N

N

N N

NH2

N

N N

N O

EtO2C

 10 min 55%

Scheme 3: Reaction of imidazo[1,5-a][1,3,5]triazin-4-amine 9 with ethoxymethylene

malonate 10 fused pyrimido[1,2-a][1,3,5]-triazine 11

Annulation of pyrimidine to 1,3,5-triazine via base catalyzed rearrangement of

the isoxazolone substituted 1,3,5-triazine20

In this method, acetamide derivative of 4,6-diamino-2-chloro-1,3,5-triazine 12 was

reacted with isoxazolone ester 13 in ethanol Both N-acetyl groups were hydrolysed

during the reaction period (10h, 80°C) to give 14 Compound 14 underwent ready rearrangement to 7-carboxyethyl-8-hydroxy-6-oxo derivative (15) on warming with 2.5 M NaOH (Scheme 4)

N N

NaOH/40°C

N N

N

NH2

NH2

N EtO2C

O

HO

N N

O O EtO2C +

78%

82%

Scheme 4: Base catalyzed rearrangement of ethyl

2-(4,6-diamino-1,3,5-triazin-2-yl)-5-oxo-2,5-dihydroisoxazole-4-carboxylate 14 to give pyrimido[1,2-a][1,3,5]-triazine

15

In this reaction, two different regioisomeric products might result via oxazolone ring

opening and subsequent pyrimidine ring annulation of 14 However, since the chosen substrate 12 for the reaction had a plane of symmetry, there was no regioselectivity

issue Therefore, regiochemistry of heterocyclization when unsymmetrically substituted substrates are used remains to be explored

Annulation of pyrimidine via intramolecular ring closure21-24

In an attempt to obtain 18 via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) catalyzed amide coupling of N-triazino anthranillic acid 16 with alanine, the formation of 15% of fused pyrimido[1,2-a][1,3,5]-triazine derivative 17

was also reported This formation of 17 was a result of intramolecular ring closure

leading to pyrimidine ring annulation which happened prior to coupling (Scheme 5) This kind of intramolecular ring closure was reported by many authors for the synthesis of various benzofused pyrimido[1,2-a][1,3,5]triazines Polyfused

pyrimido[1,2-a][1,3,5]triazine 20 was also synthesized by similar intramolecular ring closure of N-triazino anthranillic acid substrate 19 21 (Scheme 6)

18

N N N N OMe

N H

O

+ Alanine Bz, EDC.HCl, HOBt, TEA, DMF

N N

N N OMe

HN

CO 2 Bn Me

N N N O

CH3N

HN N

N N

Scheme 6: Intra-molecular ring closure of pentazacycl[3.3.3]azine 19

Refluxing 2,4-bis(o-carbomethoxyanilino)-6-dimethylamino-1,3,5-triazine 21 in

acidic condition for 16h yielded

2(4)-anilino-4(2)hydroxy-6H-1,3,5-triazino[2,1-b]quinazolin-6-one (22) Cyclization-deamination and decarboxylation were believed

to have happened in the same step These compounds were patented as peripheral vascular dilating agents as well as for the preparation of dyestuff material22 (Scheme 7)

22 21

N N

N N

OH

N H

O GAA/propionic acid, reflux, 16h

22'

N N

N N

HN O

OH

Scheme 7: Intra-molecular ring closure of 2,4-bis(o-carbomethoxyanilino)-6-dimethyl

amino-1,3,5-triazine 21

N,N-diallylmelamine or N-alkyl-N-allylmelamine 23 would undergo (6+0)

intramolecular heterocyclization to give 24 (Scheme 8) These compounds were

reported to depress CNS and were patented for tranquilizing property23,24

N

H2N

N N

N R OH

R = CH3, -(CH2)nCH3, n= 1-7, -CH2CH=CH2

23

24

Scheme 8: (6+0) intramolecular heterocyclization of N-alkyl-N’-allylmelamine 23

1.1.2 Synthesis by annulation of 1,3,5-triazine ring onto a pyrimidine scaffold

Appropriately substituted pyrimidines may also serve as starting material for the synthesis of fused 1,3,5-triazine heterocycles The desired fused 1,3,5-triazine scaffold can be formed by annulation reaction on pyrimidine This approach has been

adopted extensively for the preparation of pyrimido[1,2-a][1,3,5]triazines Suitably

functionalized 2-amino pyrimidines can be cyclized to generate

pyrimido[1,2-a][1,3,5]triazines according to the strategies described below (Scheme 9)

+ + +

C N C

This synthetic approach provides an excellent opportunity for the preparation of a

variety of functionalized pyrimido[1,2-a][1,3,5]triazines using 2-aminopyrimidines

The triatomic C-N-C synthons used in the cyclization determine the substitution

pattern at the positions 2 and 4 of the formed 1,3,5-triazine ring N-cyano-imidates 25,

isocyanates 38/46 or isothiocyanates 50 are the examples of this type of synthons

found in the literature for the construction of the desired scaffold (Fig 5) It should be noted that the regiochemistry of the ring closure was not always unambiguous and the structure assignments were often inadequate and therefore would require further verification

N RO

X = Cl, Y = O, Chlorocarbonyl isocyanate 38

X = OPh, Y = O, Phenoxycarbonyl isocyanate 46

X = Ar, Y = S, Benzoyl isothiocyanate 50

Fig 5 Examples of C-N-C synthons used in the annulation of 1,3,5-triazine onto a

pyrimidine

Annulation of 1,3,5-triazine ring with methyl N-cyanoformimidate 25a as C-N-C

fragment introducing reagent

The reaction of substituted 2-amino pyrimidin-4-ones 26 with methyl cyanoformimidate 25a in the presence of sodium methoxide could proceed via two

N-possible modes of condensation-cyclization (N2-1 vs N2-3) with 27 being the possible

intermediate 4-amino-8-oxo-8H-pyrimido[1,2-a][1,3,5]triazine (28) was formed via

N2-1 by reaction in HMPA (hexamethyl phosphoramide) at 50-55°C25 The basic

hydrolysis of 28 using 5% aqueous NaHCO3 yielded 29 whose crystal structure (as

triflate (CF3SO3-) salt) was also reported (Scheme 10) The salt formation occurred, surprisingly, at the bridgehead nitrogen26 At low temperature (20°C), 2-amino-6-methyl- 4-oxo-pyrimidine gave N2-3 cyclized product 30 regioselectively in anhydrous methanol The structure of this regioisomer 30 could be easily confirmed

as two clearly differentiated N-H signals were observed in 1H NMR analysis because

of the non-equivalence of the hydrogens of NH2 due to intra-molecular H-bonding

Conversion of regioisomer 30 to 31 was also achieved by heating in dry dimethyl

formamide (DMF) at 120°C for 2h It is important to note here that the 1,3,5-triazine ring undergoes a hydrolytic ring opening step in preference to the pyrimidine ring in

dimethyl formamide (DMF) Isomers 32 and 33 were not observed This is consistent

with the mechanism and is expected due to higher nucleophilicity of exocyclic nitrogen atom of 2-amino pyrimidin-4-one

The authors demonstrated that regioisomeric 4-aminopyrimido[1,2-a][1,3,5]triazines can be exclusively isolated by controlling the reaction conditions (viz lowering

temperature, solvent change) This opened up the possibility of interconversion

among the regioisomers of pyrimido[1,2-a][1,3,5]triazinone

N N O

N N

H 2 N R

R DMF, 120°C, 2h

R = H, Me

NaHCO 3

N N O

N N

H 2 N NaOMe, HMPA, 55°C, 19h

N

O N

HN NH 2

33 32

29

31 30

35b and 35c (N-annelated pro-drug of acyclovir) were also studied by the same

research group27,28 (Scheme 11)

N

CN + OMe

N N N N O

N N

NH2NH

N N

35c (2-hydroxyethoxy)methyl anhyd DMSO, 55°C, 7h 86

Scheme 11: Reaction of guanosine with methyl N-cyanoformimidate to give imidazo

fused pyrimido[1,2-a][1,3,5]triazines

Ceder and co-workers also reported blue coloured 1,3,4-triazacycl[3.3.3]azine 37

when 2 mol of ethyl N-cyanoformimidate 25b was reacted with

2-amino-6-methyl-pyridine 36 (Scheme 12)29

N CN +

OEt 140°C, 4h

37

Scheme 12: Reaction of 2-amino-6-methylpyridine 36 derivative with 2 moles of

ethyl N-cyanoformimidate 25b yielded fused pyrimido[1,2-a][1,3,5]triazine 37

Annulation of triazine ring using various isocyanates as C-N-C fragment introducing reagents

i) N-chlorocarbonyl isocyanate

The reactions of 2-amino pyrimidines with N-chlorocarbonyl isocyanate (38)

provided an accessible source for the preparation of 2,4-dioxo

pyrimido[1,2-a]-1,3,5-triazines introducing -C(=O)-NH-C(=O)- fragment

N

N NH2

Cl O

N C O

N N

NH N

O

O

1 3 5

2H-product was reported when 38 reacted with substituted quinazolines 41 and authors

claimed that 1H triazino[1,2-a]quinazoline-1,3(2H)-dione 42 was the product30,31

However, the possibility of the formation of

6-phenyl-3H-[1,3,5]triazino[2,1-b]quinazoline-2,4-dione 42’ as a possible product (Scheme 14) could not be ruled out

Further investigation of regioselectivity is needed for the product confirmation

Cl O

N C O

N

NH O O

N

N X

NH2

N

N X

N H

However, Nagai and coworkers32 also reported a single product formation, which they

claimed to be 6-phenyl-3H-[1,3,5]triazino[2,1-b]quinazoline-2,4-dione (44) when

reaction with amino quinazoline derivative 43 was carried out using the same reagent under identical conditions (Scheme 15) CNS stimulant activity of this product 44 was

also reported by the authors Further investigation is required whether the product

obtained was quinazolo[2,1-b][1,3,5]triazine or quinazolo[1,2-a][1,3,5]triazine

Cl O

N C O TEA, CH2Cl2, 0°C to r.t., 4h

38

N

N

NH2+

NH O O

N N

N H

N O O

pyrimido[1,2-a][1,3,5]triazin-2,4,8(3H,9H)-trione 45 (and not the other regioisomer

45’) was exclusively formed in the reaction with 2-amino pyrimid-4-one 26a (R = H)

under similar conditions (Scheme 16)

Cl O

N C

N H

NH N

N H

N O O

8

91%

26a

O

Scheme 16: Reaction of 2-amino pyrimid-4-one 26a with chlorocarbonyl isocyanate

38 to give pyrimido[1,2-a][1,3,5]triazin-2,4,8-trione regioisomer 45

ii) Phenoxycarbonyl isocyanate / Phenoxycarbonyl isothiocyanate (46)34

The formation of mesoionic

1-alkyl-4-oxo(thio)pyrimido[1,2-a][1,3,5]triazin-1-ium-2-olates (47) was reported using phenoxycarbonyl isocyanate (46a) /

phenoxycarbonyl isothiocyanate (46b) with N-alkylamino pyrimidines 39 via 3+3

cyclocondensation These mesoionic compounds were also formed by intramolecular

heterocyclization of 48 and 49 (Scheme 17) 4-oxo or 4-thioxo derivatives of

mesoionic pyrimido[1,2-a][1,3,5]triazines can be synthesized via this method

R2 = OEt

R 2 O

N X

R 1



X=O, R1 = Me, 6h, 87.5% 47a

O +

Scheme 17: Reaction of phenoxycarbonyl iso(thio)cyanate 46 with N-alkylamino

pyrimidines 39 to yield mesoionic pyrimido[1,2-a][1,3,5]triazines

iii) Benzoyl isothiocyanate (50)

The product of the reaction of diaryl amino pyrimidine derivatives 51 with nitrobenzoylisothiocyanate 50a in dry acetone was reported to be 52 (yield 12%) Again the possibility of formation of other regioisomeric product 52’ was over-looked

p-by Mahmoud and coworkers35 The spectral data provided were limited and not

sufficient for conclusive structural assignment among 52 and 52’ (Scheme 18)

Ar 1

Ar2N S

NO2

Scheme 18: Reaction of benzoyl isothiocyanate 50a with substituted 2-amino

pyrimidines 51 to give pyrimido[1,2-a][1,3,5]triazin-2-thione

B) Two bond formation (4+2) through cyclization of pyrimidine having N-C appendage at C2 with reagents introducing C-N fragment

The use of aryl isocyanates as source of C-N fragment

2,4-dioxopyrimido[1,2-a][1,3,5]triazines 40 could also be synthesized from the reaction of N,N-dimethyl-N’-(pyrimidin-2-yl)formamidine 53 with aryl isocyanates

54, giving analogous 2,4-dioxo[1,3,5]triazine product 40 in good yields36

N

53

H N(CH3)2

+ RNCO

R = Ph, 45 min, 95%

R = Ph (4-Cl), 30 min 98%

N N

N

N O O

N,N-dimethyl-N’-(pyrimidin-2-The reaction of 2-chloropyrimidine derivative 55 with ammonium thiocyanate (1 eq.)

for 1 hour followed by the addition of phenylisocyanate (1 eq.) afforded

pyrimido[1,2-a][1,3,5]triazine (57) (Scheme 20) Again, the authors provided

insufficient spectroscopic evidence in support of the product formation as well as for the regioselectivity of ring closure37 Only 2-thioxo-4-oxo-1,3,5-triazine derivatives of

the desired bicyclic heterocycle were synthesized using this method

N N

Ar

N N N N

Ar Ph

Ph S O

Scheme 20: Reaction of chloropyrimidine 55 with ammonium thiocyanate followed

by phenylisocyanate 54a gave 2-thioxo-4-oxopyrimido[1,2-a][1,3,5]triazine 57

Two-bond formation (4+2) through concurrent annulation of triazine and additional pyrimidine ring onto pyrimidine having N-C appendage at C2 with C-

N fragment introduced by CN grp of 2-cyanoacrylate 38

The process of condensation of 2,4-diaminopyrimidine 58 with 1 mole of cyano-3-ethoxyacrylate 59 in benzene which was followed by acetylation gave the product 60 Concurrent annulation of pyrimidine and 1,3,5-triazine resulted in two bond formation leading to 1,3,4,7-tetraazacycl[3.3.3]azine 62 (Scheme 21)

N N

62

NH2

N N N H

60

NH 2

COOEt N

N N

N H

HN

COOEt N

O Me

Me

COOEt p-TSA, Ph2O, 250°C, 15 min

Scheme 21: Two-bond formation (4+2) through concurrent annulation of triazine and additional pyrimidine ring onto pyrimidine having N-C appendage at C2

C) Three bond formation (2+2+2) through cyclization of pyrimidine two N-C fragments provided by 2 moles of isocyanates or isothiocyanates

The use of aryl isocyanates for pyrimido[1,2-a][1,3,5]triazine formation was first

reported by Richter39 and the approach has been continually exploited since The

product of the reaction of pyrrolo[1,2-a]pyrimidine 63 with aryl isocyanate was

claimed to be 64 (Scheme 23)

N N

64

Benzene

R

N O

O R

54

Scheme 22: Reaction of aryl isocyanates 54 with pyrrolo[1,2-a]pyrimidine 63 to give

fused pyrimido[1,2-a][1,3,5]triazindione (64)

Subsequently, Sawada et al.40 reported the formation of

pyrimido[1,2-a]-1,3,5-triazin-2,4,8-triones (67) along with their 5-methyl carbamoyl adducts (68) which were

produced with a third molecule of isocyanate (used in excess equivalent) (Scheme

23) Analogous to pyrimido[1,2-a][1,3,5]triazines,

quinazolino[1,2-a][1,3,5]triazine-1,3,6-trione (1:2 adduct similar to 67) and adducts similar to 66 and 68 were obtained

in 32%, 2% and 14% respectively They also reported that the cyclization occurred only at C-2 (not at the C-6) of the 4-pyrimidinone which may be due to electron deficiency at C-2

N

NH

O

N N O

H Me

N NH O

base

68, 25% 66

65

2 6

Scheme 23: Reaction of 4-pyrimidinone 65 with methyl isocyanate 54d to give

pyrimido[1,2-a][1,3,5]triazintrione 67

Similar 2+2+2 cycloadduct- [1,3,5]triazino[1,2-a]-quinazoline-1,3(2H)-dione 70 41

was reported to be formed by the reaction of 3,4-dihydroquinazolines 69 with phenyl isocyanate 54a at 80°C (Scheme 24) Spiro compound 70’ was never isolated

Kinetically and thermodynamically favourable cycloaddition products (possible intermediates) were also isolated and characterised using NMR and X-ray crystallography in this detailed study

N

N R'R

+

N N

N N

Ph

Ph H

N

N R'R

HN O Ph

PhN NPhO O

Pyrimido[1,2-a][1,3,5]triazine-2,4(3H)-dithiones 72 (which are isosters of

pyrimido[1,2-a][1,3,5]triazine-2,4-diones) were also synthesized similarly by

H

-SR1, -H+

Scheme 25: Reaction of 2-thioalkyl-1,4,5,6-tetrahydropyrimidines 71 with iso(thio)

cyanates 54 to give pyrimido[1,2-a][1,3,5]triazindione 72

Pyrimido[1,2-a][1,3,5]triazindithiones 77 were also prepared by the reaction of three

molecules of alkyl isocyanate with dianion 74 (generated by butyl lithium) in THF at

room temperature44 Along with 77, the reaction also produced thiocarbamoyl derivative 78 and heteropentalene derivative 79

Li+

S

S N R

Li+

N S NR S

R

Li+

N N S

S R

78

+

N S

S

R N S R

76 75

74 73

D) Three-bond formation (3+2+1) through cyclization of 2-aminopyrimidines with reagents introducing C-N fragment and one carbon atom

Camus and coworkers45 reported the reaction of aryl isocyanate 54 with oxo-pyrimidine derivative 80 to yield N2-3 cyclized pyrimido[1,2-a][1,3,5]triazine 81

2-amino-4-Here the N-C fragment was provided by the first molecule of aryl isocyanate and the carbonyl fragment was provided by the second molecule of aryl isocyanate Further,

methanolic hydrolysis of 81 gave opened ureido derivative The structure of opened ureido derivative of 81 was determined by X-ray crystallography and this was claimed to be the confirmation of structure 81 “by extension” The evidence of

ring-structural confirmation provided in this report was inadequate (Scheme 27)

O

O

1 3

81 80

R = H, 80a

R = COCH3, 80b

R = COPh(4-OMe), 80c

Scheme 27: Reaction of 2-substituted-6-(diethylamino)-5-nitropyrimidin-4-one 80

with phenyl isocyanate 84a to obtain pyrimido[1,2-a][1,3,5]triazine

Under similar reaction conditions, triazino fused tricyclic nucleoside 83 was also

reported as a product formed from deoxyguanosine28 (Scheme 28)

PhNCO, NEt3, CH3CN N

N H

N

N Ph

O

OR RO

82

Scheme 28: Reaction of deoxyguanosine 82 with phenylisocyanate to yield fused

pyrimido[1,2-a][1,3,5]triazine 83

N-(2-pyrimidinyl)triphenylphosphinimine 84 has been shown to react with aryl

isocyanate both in 3+2+1 and 4+2 fashion N-(2-pyrimidinyl)triphenylphosphinimine

84 reacts with phenyl isocyanate46 in one equivalent to give similar product 86 With

1.5 equivalent of phNCO and longer reaction duration, 87 was reported as the major product along with 12% of 86 Predominant isolation of 87 can be explained by

formation of diphenyl carbodiimide with 2 mol of phenyl isocyanate in situ Low

yield (7%) of cycloadduct 87 was also reported from

N-(2-pyrimidinyl)triphenylphosphinimine 84 with diphenylcarbodiimide confirming the proposed explanation 47 (Scheme 29) The polymerization, observed during the

reaction, was a reason for the low isolated yield of 87

N PhO

O

N

N N

N PhNPh

1.5 eq., benzene, r.t., 30-40h

63%, Method A 12%, Method B

Recently, Shehata and coworkers48 have also reported tricyclic 3+2+1

cyclocondensation product (90) N-acetylated derivative 89 reacted with excess

equivalent of phenyl isocyanate 54a to give triazolo fused

pyrimido[1,2-a][1,3,5]triazindione (90) (Scheme 30)

N N NC

N H N N Ph

O Ph NC

N N Ph

PhNCO, Py, 135°C, 3h 53%

89 88

Scheme 30: Reaction of N-(8-cyano-2-phenyl[1,2,4]triazolo[1,5-f]pyrimidin-5-yl)

acetamide 89 with phenyl isocyanate 54a fused pyrimido[1,2-a][1,3,5]triazine

In contrast to benzoyl isothiocyanates (PhCONCS), benzoyl thiocyanate (PhCOSCN)

92 gave benzoylated thiourea derivative 93 with 2-amino pyrimidine derivative 91 (Scheme 31) The attempt to cyclize using chloroethylformate 94 led to debenzoylation giving pyrimidinyl thiourea 95 which readily underwent convenient heterocyclisation using orthoformate 96 and chloroethylformate 94 forming the

desired scaffold 97 and 98 in high yields49 2-thioxo or 2-thioxo-4-oxo derivatives of

pyrimido[1,2-a][1,3,5]triazines can be synthesized using this simple method

97

98

N N N CH(OEt)3, DMF, 4h

N N N NH O

CH3

H3C

ClCO2Et DMF, r.t, 5 min.

N N N

CH3

NH N

101

R1 = R2 = aliphatic; R3 = aliphatic/aromatic

Scheme 32: Mannich condensation of 2-amino pyrimidine derivative 99 yielding

tetrahydro pyrimido[1,2-a][1,3,5]triazines 101 (43 examples)

Mannich cyclocondensation of 2-aminopyrimidine derivative 99 with 2 molecules of formaldehyde and 1 molecule of aliphatic or aromatic amine 100 led to the annelation

of tetrahydro-1,3,5-triazine ring onto amino pyrimidine 50-52 (Scheme 32) The

product 101 can exist in 1H or 9H tautomeric forms The confirmation of the

tautomeric form, carried out by using NOE and selective monoalkylation at N-9, was provided much later by Lucry and coworkers in 200253 It is important to note that induction of such tetrahydrotriazine Mannich adducts with purine nucleotides

(deoxyguanosine in particular) 54,55 has been linked to carcinogenicity and mutagenicity of formaldehyde and glutaraldehyde in humans

F) Two-bond formation (5+1) through cyclization of pyrimidine having N-C-N appendage at C2 with one carbon atom

The reaction of 2-(pyrimidinylamino)benzimidazole 102 with phosgene (providing the sixth atom) 103 in the presence of triethylamine yielded the cyclized product (104) in quantitative yields (Scheme 33) The product was characterised by IR, mass

spectrometric and elemental analyses56

N N N O

Scheme 33: Reaction of 2(benzimidazol-2-yl amino)pyrimidine 102 with phosgene

103 to yield tetracyclic fused pyrimido[1,2-a][1,3,5]triazine 104

This type of 1,3,5-triazine ring annulation can also be achieved using other 1C

inserting synthons such as orthoesters 96 Derivatives of polyfused system 106

comprising pyrimido[1,2-a][1,3,5]triazine nucleus were prepared by orthoester

assisted ring closure between the amino group of pyrazoline and NH group of pyrimidine (Scheme 34)57

+ R2C(OEt)3

R1 = Me, Ph, 4-MePh

R2 = H, CH3, C2H5, Ph

N N

N N

N

R1

106 105

Scheme 34: Reaction of 2[3’-substituted-5’-aminopyrazolyl]-1,4,5,6-tetrahydro

pyrimidine 105 with orthoesters 96 fused pyrimido[1,2-a][1,3,5]triazine

Another 5+1 heterocyclization was reported by Ishikawa et al58 Pyrimidinyl

cyanamide 107 was first converted to pyrimidinyl urea intermediate 108 Further

heating of 108 at 200-220°C for 30 min gave 29% of pyrimido[1,2-a][1,3,5]

triazin-2,4-dione 110 along with tetrahydro 2-iminopyrimidine 109 Formation of 110 could

be explained on the basis of thermal disproportionation of pyrimidinyl urea

N N

H

N

N C

Bn HCl, EtOH, r.t.

O

NH2

N H

N BnNH +

O O

1.1.3 Synthesis by concurrent formation of both the pyrimidine and triazine ring

Two bond formation with concurrent cyclization using anthranillate and amidine as pyrimidine and triazine source respectively 59-61

Benzenesulfonic acid catalyzed cyclization of potassium N,N-dicyanobenzamidine

112 with methyl anthranilate 111 in methanol gave triazino[2,1-b]quinazolone 114 instead of triazino[1,2-a]quinazolone 114’ In order to confirm the structure of 114, an

independent synthesis of this compound by annulation of quinazoline onto triazine

was also performed Product 114 was obtained by initial nucleophilic displacement of monochloro-1,3,5-triazine derivative 113 by heating in glyme followed by intramolecular ring closure of the product 115 using sodium hydride (Scheme 36)

N

NH N

NH2

CO2Me

NC NN NC R

R

O

a b c

Scheme 36: Reaction of N,N-dicyanobenzamidine 112 with methyl anthranilate 111 to

yield benzofused pyrimido[1,2-a][1,3,5]triazine 114 (11 examples)

Concurrent formation of triazinone and tetrahydropyrimidine in one-pot Wittig/heterocyclization/substitution 62

Aza-Subsequent to the Aza-Wittig/heterocyclization reaction of 116 with 3-chlorophenyl isocyanate 118, a nucleophilic displacement of one of the halogen atoms would lead

to further tetrahydropyrimidine annulation forming tetracyclic

tetrahydropyrimido-1,3,5-triazinobenzimidazolium salts 119 or/and 119’ The formation of 2 regioisomers

via ring closure route a or b was possible (Scheme 37) 13C HMBC spectrum, which allows detection of a correlation between a proton and the carbon atoms two and three bonds away, was used to confirm the predominant regioisomer 12-substituted 9-(aminocarbonyl)-1-(3-chloropropyl)-6-oxo-1,2,3,4-tetrahydropyrimido[2’,1’:4,5]

[1,3,5]triazino-[1,2-a]benzimidazol-12-ium trifluoroacetate 120 (93-96%) As only in

120, and not 120’, H-2 and the α-CH2 expected to show an easily observable long

range correlation (3J)

N N

N N

R N

Cl

N

N N

N N

R N

N

N N PPh3

R N

N N

12a 11 7 5

R N

N

N N N

CF 3 COO

118 117

Derivatives of pyrimido fused bicyclic heterocyclic scaffolds are found in numerous

anticancer agents (Fig 6) Members of pyrazolo[1,5-a]pyrimidine63,64,

pyrrolo[3,2-d]pyrimidine65 (where pyrimidine is fused to five-membered rings like pyrrole and pyrazole) scaffold have been reported as KDR kinase, Btk kinase and CDK-2

inhibitors Benzo[d]pyrimidine or quinazoline66-68 derivatives such as Imatinib, Canertinib, Lapatinib, Vandetanib, Tomudex (plevitrexed), Enastron, Vanastrol possess antitumor properties via different mechanisms like tyrosine kinase, Vascular endothelial growth factor (VEGF), thymidylate synthase (TS) and human kinesin Eg5

inhibition Pyrazino[1,2-a]pyrimidine69,70 derivatives have been patented as catenin pathway signaling modulators whereas numerous derivatives of the

Wnt/β-pyrido[2,3-d]pyrimidine71 and pyrimido[1,2-a]pyrimidine72 scaffold are tightly covered in the kinase patent space

N N N

N N

N

N N

pyrimido[5,4-e][1,2,4]triazine

scaffold

N

N N

H O

CDK4 + CDK6 inhibitor

Pfizer (Phase II)

N N NH

N N

N N N quinazoline

pyrazino[1,2-a]pyrimidine scaffold

PD-0332991

pyrimido[1,2-a][1,3,5]triazine

scaffold

N N N

O O

H

H HO

Wnt/-catenin pathway modulator

Fig 6 Structurally similar N-containing bicyclic heterocyclic scaffolds

Pyrimido[5,4-e][1,2,4] triazine or 7-azalumazine73,74 scaffold (where pyrimidine is fused to 1,2,4-triazine ring) is present in natural antitumor antibiotic drug-reumycin

(6-Methylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione) and its derivatives –

fervenulin, MSD-92 and toxoflavin

Due to the close structural similarity with the above pyrimido fused bicyclic scaffolds,

the derivatives of pyrimido[1,2-a][1,3,5]triazine scaffold are anticipated to possess

anticancer property So far, very few reports on the biological activity from this class are available in literature Anti-microbial properties of positional isomer-

pyrimido[1,2-a][1,3,5] triazine derivatives 97 and 98 were explored by Abou Dobara

and coworkers49 (Fig 7)

97 98

N

N N NH O

S

CH3

H3C N

N N

N S

CH3

H3C

Fig 7 Antimicrobial pyrimido[1,2-a][1,3,5]triazine derivatives

Anti-fungal activity was stated for derivatives 101a against Microsporum canis and

average affinity of 101b for serotoninergic 5-HT1A and 5-HT2B receptors was also published by Lucry and coworkers53

N

N N N O

Fig 8 Antimicrobial pyrimido[1,2-a][1,3,5]triazines

In addition to antimicrobial activity, weak anti-inflammatory activity57 for tricyclic

3+2+1 cyclocondensation product (90), anti-hypertensive activity for 126 and 127 as well as CNS depressant activity for 2424 and CNS stimulant activity of 1721 were also reported from the scaffold (Fig 9-11)

N N N O

Ph

CN

N N Ph

90

Fig 9 Anti-inflammatory pyrimido[1,2-a][1,3,5]triazine derivative 90

N H

N N

N NHPh O

O

N H

N N

N O

NHPh O

Fig 10 Anti-hypertensive pyrimido[1,2-a][1,3,5]triazine derivatives 126 and 127

N N

N N

OMe

N H H

O

CO2Bn

Me N

N N N

NH2

NH2HO

24

Fig 11 CNS active pyrimido[1,2-a][1,3,5]triazine derivatives 24 and 17

It was reasonable to expect and explore antitumor properties of the

pyrimido[1,2-a][1,3,5]triazine scaffold due to its similarity with various scaffolds described in Fig

6 However, no report was available regarding the antiproliferative activity of the

scaffold Antiproliferative activity of this scaffold was first reported by our group75

where 125 was found to be a promising lead with GI50 of 17 µM and 15 µM against

MDA-MB-231 and A549 cancer cell lines respectively (Fig 12)

N

N N

NH

NH2Br

125

Fig 12 Antiproliferative pyrimido[1,2-a][1,3,5]triazines.

1.2 Research Hypothesis and Objectives

An overview of a variety of methods for the preparation of

pyrimido[1,2-a][1,3,5]triazine as well as its derivatives where fused ring systems are incorporated

into this scaffold has been presented In terms of practicality, the construction of the 1,3,5-triazine ring onto a pyrimidine skeleton has been shown to be the most versatile Regardless of the synthetic approaches used for the preparation of pyrimido[1,2-

a][1,3,5]triazines, the ring closure on an existing ring that is unsymmetrically

substituted can lead to the formation of either one regioisomer or both regioisomers as products Therefore, the correct structural characterization of the regioisomer is important In fact, many reports have not even discussed the regiochemistry, and the evidence for structural confirmation were often insufficient and inconclusive However, there are a few reports (from Leonard’s group) that studied regioselectivity

in detail and pointed out that the careful control of reaction conditions might influence the regiochemistry, thereby favouring the generation of preferred regioisomers Most

of the synthetic approaches shown in previous sections could not provide the flexibility of different substitution at various positions around the fused ring, and careful evaluation of the total literature suggested that most authors largely focussed

on the formation of dioxo/dithio/oxothiooxo derivatives of the scaffold Moreover, the reported yields were sometimes low In some cases, the reagent like phosgene were used; this made the procedure hazardous Most starting materials (like isoxazolone in scheme 4) are not commercially available, and non reproducibility of the reported

procedures made the access to pyrimido[1,2-a][1,3,5]triazine derivatives difficult

Therefore, there is a definite need to find more practical approaches for the synthesis

of these pyrimido[1,2-a][1,3,5]triazines Very few reports on the biological activity of

this class of compounds, as concluded from the review, have left the area open for further investigation In particular, no report of antiproliferative activity has been reported except from our laboratory Since antiproliferative activity of monocyclic 1,3,5-triazines has been repeatedly demonstrated by our group and others, it would be reasonable to hypothesize that the exploration of fused 1,3,5-triazines, in particular

pyrimido[1,2-a][1,3,5]triazines, could generate some antiproliferative leads

For over a decade, our laboratory has focused its research activities on the core structure of 1,3,5-triazine76-83 where particular interest has been aimed at exploring the anticancer properties of its derivatives In continuation of the efforts of our

research group to find new anticancer leads, the interest has been extended to include

the pyrimido[1,2-a][1,3,5]triazine system and its benzofused analogues with the

hypothesis that appropriate substitutions around this heterocyclic system would generate potential antiproliferative leads

Therefore, the project was undertaken with the following objectives:

1 To develop new and practical synthetic methodologies for

pyrimido[1,2-a][1,3,5]triazine and its benzofused analogues The approach selected for the

synthesis of the desired scaffold was to annulate the 1,3,5-triazine ring onto a pyrimidine ring In particular, the 5+1 cyclocondensation (refer 1.2.6) of appropriate azaheteroarylguanidines using a variety of one carbon inserting reagents such as aldehydes or orthoesters would be undertaken As evident from the literature review, this approach for the synthesis of pyrimido[1,2-

a][1,3,5]triazines has not been explored In cases where two different

regioisomers could result during the formation of the desired products, further investigation would be conducted to understand the regiochemistry of the process

2 To evaluate the synthesized compounds for antiproliferative activity against the A549 (lung cancer), MDA-MB-231 (breast cancer) and MRC-5 (normal fibroblast) cell lines and to identify potential leads Elucidation of the structural activity relationships (SAR) for the synthesized compounds would be attempted

3 To investigate the mechanism of the induced cytotoxicity by the synthesized lead compounds obtained from various structural modifications In particular, virtual screening methods would be exploited for prediction of putative targets for the identified lead compounds

CHAPTER 2 PYRIMIDO[1,2-a][1,3,5]TRIAZIN-6-ONES AS PROMISING

NEW SCAFFOLD FOR ANTIPROLIFERATIVE ACTIVITY

In this chapter, the synthesis of the scaffold pyrimido[1,2-a][1,3,5]triazines will be

described Initially, the aim was to introduce a basic group in position 2, an alkyl or aryl group at position 4, a keto group at position 6 and an alkyl group in position 8 In order to achieve this, 1,3,5-triazine ring annulation onto a pyrimidine scaffold approach via 5+1 cyclocondensation was adopted (Fig 13) The penta atomic synthon for achieving this cyclocondensation was provided by 1,6-dihydropyrimidin-2-yl guanidine The synthesis of this versatile synthon is described below

N

N N N

1 2

3 4 5 6 7 8 9

ƒ

N

N N

N + C

The required intermediate: N-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)guanidine

(128) was prepared from biguanide 132 Biguanide 132 (as sulfate salt) was prepared

from cyanoguanidine according to a reported procedure by Uohama et al.84 Since

unsubstituted and N-substituted biguanides are polynucleophiles that may cyclize by

fragment A (penta atomic) or by fragment B (triatomic) depending on the reagent used

(Scheme 38), it was necessary to have an overview of the reactions of biguanides with different carbonyl compounds

The reaction of unsubstituted biguanide (132a, R1, R2 = H) with ethyl cyanoacetate

and ethyl malonamide furnished 2,4-diamino-1,3,5-triazines 133a (R3 = CH2CN, R4 =

H) and 133b (R3 = CH2CONH2, R4 = H)85 Biguanides (132b R1, R2 = aliphatic / 132c

R1 = H, R2 = aryl) react with aromatic and aliphatic esters86,87 to give

2-amino-1,3,5-triazines 133c-i (R3 = CH2Cl, COOK, Py, 4-OH-3,5-t-Bu2C6H2, CF3, Me; R4 = H, Ph,

4-OH-3,5-t-Bu2C6H2) By reacting ketones with alkoxybiguanides (R1 = H, R2 =

OCH2OPh, 132d; R1 = H, R2 = OCH2CH2OPh, 132e), Jensen et al88 have prepared

2,4-diamino-4,6-dihydro-1,3,5-triazines 134a (n = 1) and 134b (n = 2) With the above substrates, biguanides 132 act as penta atomic synthons

2 R 1 RN

N N H

N H

N N H

NH

NH2

O

Ph Ph

CH3

N H

N N H

NH NH

O Ph Ph

CH3+

N

N N

NH N

Scheme 38: Summary of reaction of biguanides 132

However, if the ester molecule has a β-carbonyl group, the cyclization takes a different

direction where the biguanide behaves as a triatomic synthon, and the reaction product

was assigned the structure of 4-hydroxy pyrimidine 13689 Similarly, the reaction of

compound 132a with benzoin leading to 2-guanidino imidazole 13590 has been

described as well as the reaction of p-tolylbiguanide 132c (R = p-tol) with benzil

yielded a mixture of regioisomers 135a and 135b91 due to cyclization on different

sides of the triatomic synthon 132

In the present context, the cyclocondensation of biguanide and its analogues 132 with ethyl acetoacetate gave 128-131 as reported by Curd and Rose92 (Scheme 39, Method A) There was a need to develop an alternative method of obtaining substituted

dihydropyrimidin-2-yl)guanidines 128-131 as the obtained yields of biguanides

132a-d synthesize132a-d by Cur132a-d an132a-d Rose’s metho132a-d (i.e metho132a-d A) were poor especially for substituted guanidines 129-131 Therefore, dihydropyrimidin-2-yl guanidines (128-

131) were synthesized via microwave (MW) assisted nucleophilic addition of amines

protic acid or TMSCl catalyzed conditions (method B, scheme 39) In both of these conditions, the reaction occurring inside the vessel was the same However in the latter case, the function of TMSCl was to act as a source of anhydrous HCl as a result of its water scavenging property (Scheme 40) The reaction times were shorter, workup was easy, obviating the need of column chromatography, and appreciable yields (36-93%) were obtained with a variety of primary and secondary amines with alkyl, aryl and aralkyl substituents Method B was found to be more versatile as a variety of N’-

substituted pyrimidinyl guanidines in addition to 128-131 were also synthesized

(discussed later) and therefore was robust for molecular library generation Moreover

there was no possibility of obtaining a mixture of regioisomer (as in the case of 135a and 135b) when unsymmetrical biguanide (R1=H, R2=Ar/alkyl) was used as a

substrate

H3C N

NH N

iii

ii i

Scheme 39: Synthesis of

1-substituted-3-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)guanidines 128-131 Reagents and conditions i) Ethyl acetoacetate, 40% NaOH

(aq), EtOH rt; ii) NH4Cl, C6H5OH, 120°C or morpholine HCl, butanol, reflux; iii)

NR1R2 HCl, MW, 160°C, 15 min or NR1R2, TMSCl, MeCN, 12 min., 160°C followed

by iPrOH, 125°C, 30 sec

Scheme 40: Water scavenger trimethylsilylchloride 140

The guanidine group in 128-131 provided the penta atomic synthon for 1,3,5-triazine

ring annulation to synthesize the desired pyrimido[1,3,5]triazines

2.1.1 Tautomerism in N-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)guanidine

As shown in a detailed research on the N3H ↔N1

H amide-amide tautomerism of pyrimidones93, the preferred structure is 128 and not the hydroxy tautomer 142 or the

4-1H tautomer 143 (1,4-dihydro form) In this work, the 3H tautomeric form was

assigned to pyrimidine in the products of cyclization of biguanides with β-keto ester

despite the assignment of hydroxy tautomer 142 by almost all the authors The

existence of pyrimidinones in the 3H form might be attributed to a smaller dipole moment of this tautomer than the 1H tautomer94 The observation of broad signals of the guanidine groups in 1H NMR spectrum of 128-131 suggested the possibility of

guanidine tautomerism (Fig 14) The singlet at 10.58-11.52 ppm was assigned to the pyrimidine N1H group and δ 5.45-5.58 ppm to C5H

NH

NH2O

NH N

NH2O

NH N

NH O

128a

3 1

N N

NH2OH

4-one

Fig 14 Tautomerism of 4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl) guanidine

2.2 Synthesis of 2-aminopyrimido[1,2-a][1,3,5]triazin-6-ones with substituent

variation at position 2 of the ring

Variation of the amino group at position 2 was first used as an exploratory strategy to determine whether it can potentiate the antiproliferative activity of the synthesized

pyrimido[1,a][1,3,5]triazin-6-ones A morpholine ring was also included as a

2-amino group replacement due to a number of reasons Morpholine motif is a common pharmacophoric group present in many inhibitors of phosphatidylinositol 3 kinase and phosphatidylinositol 3 kinase like kinases such as ZSTK 474 95-103 The compounds having one or more morpholine ring/s attached to 1,3,5-triazine ring have been reported to be potent and selective inhibitors of mammalian target of rapamycin (mTOR)104 As illustrated in Figure 15, some of these compounds are 144 (IC50(human

bladder cancer cell, 5637) = 12.9μM)105

and 145 (IC50(Colon Cancer COLO 205) =

2.1nM)106 Since morpholine is basic, bigger in size than the amino group and has ‘O’

as an acceptor, it also allows us to investigate the effect of attaching a bulkier group (with an H-bond acceptor) at position 2 on the antiproliferative activity

N N

N N N

N

N F F

ZSTK 474

N N

N N

H2N

O N

144

N N

N N

Fig 15 Morpholinyl analogues that showed antiproliferative activity

The reaction of 128-131 with various aldehydes was carried out either in acetic acid or

ethanol with piperidine as the catalyst, to give four libraries of

4-(het)aryl-8-methyl-4,6-dihydro-1(3)(9)H-pyrimido[1,2-a][1,3,5]triazin-6-ones substituted at position 2

and position 4 The yields obtained for the first library (149a-l) having a free amino

group (i.e NR1R2 = NH2) were in the range of 52-74% Substitution at position 4 was achieved using various aryl substituted aldehydes guided by Criag plot107, 5/6 membered heteroaryl (furyl, thiophenyl, pyridyl), carbocyclic (cyclohexyl), aralkyl (R4

= CH2CH2Ph), alkyl (R = Me) The range of substituents were covered at position 4 in

order to demonstrate the robustness of the method (Scheme 41) The yields obtained

for the second (150a-g; NR1R2 = NHCH3), third (151a-j; NR1R2 = N(CH3)2) and

fourth library (152a-j); NR1R2 = morpholino) were in the range of 46-58%, 55-79% and 53-94% respectively The substituents at position 4 were kept the same in order to facilitate the comparison among different libraries for the elucidation of SAR The solubility of the compounds, both in water and in organic solvents, were greatly

enhanced by the incorporation of a morpholine ring in the fourth library (152a-j)

Scheme 41: Synthesis of

2-substituted-4-het(aryl)-8-methyl-3,4-dihydropyrimido[1,2-a][1,3,5] triazin-6-ones (149-152)

The structure of the products was established using NMR spectroscopy The formation

of the dihydro-1,3,5-triazine ring in the reaction was suggested by the signal at

55.3-61.6 ppm in the 13C NMR spectrum of the product This strong evidence of sp3hybridization at C-4 ruled out the formation of the possible Schiff base-like compound

147 (Scheme 41) The absence of cross peaks between signals of protons located at sp3

hybridized carbon and the methyl group in pyrimidine in the 2D NOESY experiment

excluded possible formation of structure 148 and gave evidence of regioselectivity of

the ring closure Hence, the structure of

2-amino-4-(het)aryl-4,6-dihydro-1(3)(9)H-pyrimido[1,2-a][1,3,5]triazin-6-one (149-152) has been assigned for the compound

obtained

2.2.1 Prototropic tautomerism in

8-methyl-4-het(aryl)-3,4-dihydro-pyrimido[1,2-a][1,3,5]triazin-6-one

Theoretically, structure 149 may exist in four tautomeric forms: 1H- (B), 3H- (A), 9H-

(C) and 6-hydroxy- (D) tautomers (Fig 16) It was of interest to investigate which of

the forms exists in solution

H3C N

H

N N N O

NH2

R4

1 3

H3C N

N N H N O

NH2

R4

9

Fig 16 2-Amino-8-methyl-4-het(aryl)-3,4-dihydro-pyrimido[1,2-a][1,3,5]triazin-6-one

(A) and its possible tautomeric forms (B-D)

In a 2D NOESY NMR experiment, the signal of the migrating proton gave cross peaks

with the singlet of H-4 and doublet of protons at ortho-position of phenyl ring thereby

suggesting the 3H- form (A) to be predominant in Me2SO-d6 solution (Fig 16) Cross

peaks were observed neither between the migrating proton and methyl protons nor between the migrating proton and H-7, further excluding the possibility of the existence of other forms Moreover, splitting of the signals of N(3)H and proton at sp3

hybridised carbon of triazine ring (J = 0 - 3.0 Hz) further confirms the migrating

proton in proposed tautomeric forms is localised adjacent to the hydrogen of sp3hybridised carbon

Next, using theoretical calculations, we tried to assess which of the forms were energetically more favourable For the tautomeric study, 4-phenyl-8-methyl analog

(149a) was used as a model compound

2.2.2 Ab-initio and DFT calculations:

Electronic structure methods use the laws of quantum mechanics rather than classical physics as the basis for their computations Quantum mechanics states that the energy and other related properties of a molecule may be obtained by solving the Schrodinger equation;

HΨ= EΨ

where h = Planck’s constant; Ψ = wave function and H = Hamiltonian operator

The Schrödinger equation is a many-body problem, whose computational complexity grows exponentially with the number of electrons, and hence, exact solutions to the Schrödinger equation are not computationally practical except for the smallest systems Electronic structure methods are characterized by their various mathematical approximations to its solutions There are two major classes of electronic structure methods:

1) Semi-empirical methods, such as AMI, MINDO/3 and PM3, implemented in programs like MOPAC, AMPAC, Hyperchem and Gaussian use parameters derived from experimental data to simplify the computation They solve an approximate form of the Schrödinger equation that depends on having appropriate parameters available for the type of chemical system under investigation Different semi-empirical method are largely characterized by their different parameter sets

2) Ab-initio methods, unlike either molecular mechanics or semi-empirical

methods, use no experimental parameters in their computations Instead, their computations are based solely on the laws of quantum mechanics-the first

principle referred to in the name ab initio- and on the values of a small number

of physical constants: the speed of light; the masses and charges of electron

nuclei; Planck’s constant Ab-initio methods compute solutions to the

Schrodinger equation using a series of rigorous mathematical approximations

Semi-empirical and ab-initio differ in the trade off made between computational cost

and accuracy of results Semi-empirical calculations are relatively inexpensive and

provide reasonable qualitative description of molecular systems In contrast, ab-initio

computations provide high quality quantitative predictions for a broad range of systems They are not limited to any specific class of system However, commonly

used ab-initio methods like Hartree- Fock theory does not include a full treatment of

the full effects of electron correlation (the energy contributions arising from electrons interacting with one another) For systems and situations where such effects are important, Hartree-Fock results may not be satisfactory Therefore, within the last decade, density functional theory (DFT) has offered a computationally less expensive

yet reasonably accurate alternative to ab-initio methods for including correlation

corrections in calculating molecular properties such as geometries, frequencies and energies Density functional theory methods include some of the effects of electron correlation much less expensively than traditional correlated methods DFT methods

compute electron correlation via general functional of the electron density DFT

functionals partition the electronic energy into several components which are computed separately: the kinetic energy, the electron-nuclear interaction, the Coulomb repulsion and an exchange-correlation term accounting for the remainder of the electron-electron interaction (which is itself divided into separate exchange and correlation components) In DFT, the Schrödinger equation is reformulated to obtain the Kohn Sham equations Due to the ability to obtain good solutions to the Kohn Sham equation for systems containing tens of atoms and its applicability to address critical questions in a wide range of disciplines, the Nobel Prize was awarded to John Pople and Walter Kohn in 1998 Comprehensive review on methodology and

applications of ab-initio quantum chemistry can be obtained from ref 108 and 109

In this work, we used software called Gaussian which offers the entire range of electronic structure methods In our case, the structures of the four tautomeric forms proposed were optimized with the MP2/6-31G(d,p) basis set# Since there was no imaginary frequency in the vibrational spectra, all the tautomers were confirmed to exist at stationary points corresponding to the local minima on the potential energy surface Single point energy calculations$ at different levels of theory were then