Structure-based design, synthesis and preliminary evaluation of selective inhibitors of dihydrofolate reductase from Mycobacterium tuberculosis

Bioorganic & Medicinal Chemistry 15 (2007) 4552–4576

Structure-based design, synthesis and preliminary evaluation

of selective inhibitors of dihydrofolate reductase from

Mycobacterium tuberculosis

Mervat H. R. I. El-Hamamsy,^ꢀAnthony W. Smith,^ꢁAndrew S. Thompson

and Michael D. Threadgill^*

Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK

Received 19 December 2006; revised 2 April 2007; accepted 5 April 2007

Available online 10 April 2007

Abstract—Tuberculosis is an increasing threat, owing to the spread of AIDS and to the development of resistance of the causative

organism, Mycobacterium tuberculosis, to the currently available drugs. Dihydrofolate reductase (DHFR) is an important enzyme of

the folate cycle; inhibition of DHFR inhibits growth and causes cell death. The crystal structure of M. tuberculosis DHFR revealed a

glycerol tightly bound close to the binding site for the substrate dihydrofolate; this glycerol-binding motif is absent from the human

enzyme. A series of pyrimidine-2,4-diamines was designed with a two-carbon tether between a glycerol-mimicking triol and the

6-position of the heterocycle; these compounds also carried aryl substituents at the 5-position. These, their diastereoisomers, ana-

logues lacking two hydroxy groups and analogues lacking the two-carbon spacing linker were synthesised by acylation of the anions

derived from phenylacetonitriles with ethyl (4S,5R)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, ethyl (4S,5S)-

4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, tetrahydrooxepin-2-one and 2,3-O-isopropylidene-^D-erythronolac-

tone, respectively, to give the corresponding a-acylphenylacetonitriles. Formation of the methyl enol ethers, condensation with

guanidine and deprotection gave the pyrimidine-2,4-diamines. Preliminary assay of the abilities of these compounds to inhibit

the growth of TB5 Saccharomyces cerevisiae carrying the DHFR genes from M. tuberculosis, human and yeast indicated that 5-phe-

nyl-6-((3R,4S)-3,4,5-trihydroxypentyl)pyrimidine-2,4-diamine selectively inhibited M. tuberculosis DHFR and had little eﬀect on the

human or yeast enzymes.

1. Introduction

four months, and has a cure rate of up to 95%, given pa-

tient compliance.⁴Poor patient compliance with this

prolonged regimen, together with other factors, has led

to the emergence of multidrug-resistant tuberculosis

(MDR-TB), against which DOTS is relatively ineﬀec-

tive.^5,6In view of this, DOTS-Plus (DOTS plus sec-

ond-line TB drugs) is now recommended for treating

MDR-TB and TB in areas with high incidence of

MDR-TB.⁴However, DOTS-Plus is expensive, takes

longer to administer and has signiﬁcant side-eﬀects.⁷

Tuberculosis (TB) is responsible for the highest number

of deaths of all infectious diseases.¹Rates of TB con-

tinue to rise, leading to an estimated eight million new

cases every year and an annual death toll of two mil-

lion.²Several factors have contributed to this increase,

such as the HIV pandemic.³Current therapy (DOTS)

consists of an initial phase with four drugs, isoniazid,

rifampin, pyrazinamide and ethambutol daily for two

months, followed by a continuation phase of treatment

with isoniazid and rifampin thrice weekly for a further

Dihydrofolate reductase (DHFR) is an important en-

zyme in the folate cycle^8,9which supplies one-carbon

units, derived from the action of serine, hydroxymethyl-

transferase^10,11on L-serine, for the biosynthesis of deox-

ythymidine monophosphate (dTMP). Inhibition of the

folate cycle leads to interruption of the supply of thymi-

dine and thus to inhibition of DNA biosynthesis and

inhibition of proliferation of cells. Inhibition of prolifer-

ation is a useful goal in the therapy of cancer¹²and

of bacterial and protozoal infections.¹³Highly potent

Keywords: Mycobacterium tuberculosis; Dihydrofolate reductase;

Pyrimidine-2,4-diamine; Glycerol; Structure-based drug design.

*

Corresponding author. Tel.: +44 1 225 386 840; fax: +44 1 225 386

114; e-mail: m.d.threadgill@bath.ac.uk

ꢀ

Present address: Faculty of Pharmacy, Tanta University, Tanta,

Egypt.

ꢁ

Present address: School of Pharmacy, University of London, 29–39

Brunswick Square, London, UK.

doi:10.1016/j.bmc.2007.04.011

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4553

CO₂H

O

N

H

CO₂H

H₂N

NH₂

N

H

N

HN

H₂N

N

H

N

Me

N

H

N

1

2

Me

N

Cl

NH₂

N

NH₂

OMe

N

Cl

N

NO₂

N

H₂N

N

Et

H₂N

R

H₂N

N

Et

H₂N

N

OMe

3

a: R = Me

5

6

4

4b: R = Et

Figure 1. Structures of the DHFR substrate dihydrofolate 1 and the inhibitors methotrexate 2, pyrimethamine 3, DDMP/metoprine 4a, etoprine 4b,

methylbenzoprim 5 and trimethoprim 6.

inhibition of DHFR has been achieved with analogues

of the substrate, dihydrofolate 1 (Fig. 1). Methotrexate

2 is a highly potent inhibitor of mammalian DHFR

and mammalian tumour DHFR (IC₅₀= 2.5 nM vs. rat

liver DHFR)¹⁴and is one of the most widely used

anticancer antimetabolite drugs. It has ca. sevenfold

selectivity for inhibition of human DHFR versus

Mycobacterium tuberculosis DHFR.¹⁵

M. tuberculosis DHFR and other bacterial DHFRs,

yet it is reported to lack potency (IC₅₀16.5 lM) and

to be only ﬁve-fold selective for inhibition of M. tuber-

culosis DHFR versus the human enzyme.¹⁵There is thus

a great need for rationally designed selective inhibitors

of M. tuberculosis DHFR for treatment of this wide-

spread and often fatal disease.

The biological activities of pyrimidine-2,4-diamines

have shown that it is not necessary to have the full pte-

ridinediamine structure. These ‘non-classical’ inhibitors

have advantages in that they are more lipophilic than

2 and can enter cells by passive diﬀusion, not requiring

the folate carrier. Pyrimethamine 3 was developed over

50 years ago as a DHFR-inhibiting antimalarial drug;¹⁶

it has selectivity for inhibition of Plasmodium falciparum

DHFR activity of ca. forty-fold versus human DHFR.¹⁷

It is several orders of magnitude less potent than 2

against human DHFR.^17–19Sulfadoxine/pyrimethamine

plus isoniazid has some utility as prophylaxis against

tuberculosis in HIV-positive pateints²⁰but isoniazid it-

self has been implicated in inhibition of M. tuberculosis

DHFR after metabolism.²¹DDMP/metoprine 4a is a

close analogue of 3 which shows a similar proﬁle of inhi-

bition of DHFRs, showing some activity as an antitu-

mour agent in clinical trial.²²However, this compound

is also a highly potent inhibitor of histamine N-methyl-

transferase,^23,24leading to neurological complications

with its use. The 6-ethyl analogue etoprine 4b shows

similar antileukaemic activity;²⁵its inhibition of testicu-

lar DHFR causes infertility in male rats.²⁶Methylbenzo-

prim 5 was designed as a non-classical DHFR inhibitor

which lacks the full pteridine ring structure of metho-

trexate 2 but remains extremely potent against mamma-

lian DHFRs (IC₅₀vs rat liver DHFR 3.2 pM) with some

antitumour activity.¹⁴Interestingly, this compound is

markedly less active against Pneumocystis carinii, Toxo-

plasma gondii and Escherichia coli DHFRs;^14,18these

activities have been rationalised in a crystallographic

and modelling study.¹⁸Trimethoprim 6, in which the

5-aryl substituent is linked through a methylene bridge

for increased ﬂexibility, is often cited as an inhibitor of

2. Structure-based design

Several groups have pointed to structural diﬀerences be-

tween M. tuberculosis DHFR and human DHFR as

possible opportunities for the design of selective inhibi-

tors^15,27–29but few studies have exploited these diﬀer-

ences successfully in rational drug design for TB.³⁰Da

Cunha et al.³⁰have suggested that addition of hydro-

phobic groups to 5-deazapteridines should increase

selectivity, based on six examples. Suling et al.³¹have

achieved >100-fold selectivity for inhibition of the

Mycobaterium avium DHFR versus human DHFR

using similar 5-methyl-5-deazapteridine-2,4-diamines

but have not published results for M. tuberculosis

DHFR. Thus, the way is open for rational structure-

based design of selective inhibitors of M. tuberculosis

DHFR exploiting a major diﬀerence between human

and M. tuberculosis enzyme structures.

Li et al. reported crystal structures of M. tuberculosis

DHFR. One structure contains methotrexate 2 bound

at the dihydrofolate-binding site and NADP⁺at the

NADP⁺-binding site but also contains a glycerol tightly

bound in an adjacent pocket where it forms H-bonds

with Asp²⁷, Gln²⁸and Leu²⁴(Fig. 2a).²⁸This glycerol

is also present in the structure of M. tuberculosis DHFR

with the 1,3,5-triazine-2,4-diamine inhibitor Br-

WR99210 bound but is absent from the crystal of M.

tuberculosis DHFR containing 6, probably owing to

the fact that the trimethoxyphenyl unit causes the tri-

methoprim to bind in a diﬀerent manner, causing the

Gln²⁸side-chain to be disordered.²⁸A more detailed

examination of the environment of the glycerol reveals

additional H-bonds (Fig. 2b), as indicated by O–O and

4554

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

Figure 2. Images of structures of DHFR from M. tuberculosis, with methotrexate 2 bound at the dihydrofolate-binding site. (a) View of the structure

of M. tuberculosis DHFR with 2 bound, showing the glycerol molecule bound close to the active site (crystal structure reported by Li et al.²⁸

(glycerol and 2 are shown as rods and balls; DHFR is shown as a surface with blue cationic, red anionic and grey hydrophobic neutral). (b) Proposed

)

˚

H bonds from the bound glycerol to the residues surrounding the glycerol pocket (atoms within 3.9 A of the glycerol are shown as rods; other atoms

and bonds are shown as wires).

O–N distances and appropriate orientations. O(1)–H

makes a H-bond with the side-chain amide carbonyl

oxygen of Asp²⁷; O(1) is also involved as an acceptor

in a H-bond with the indole N–H of Trp²². O(3) is

also held in a two H-bond clamp; O(3)–H makes a

H-bond with the carbonyl oxygen of Leu²⁴and is also

an acceptor in a H-bond with the N–H of the same ami-

no acid. O(2) accepts a single H-bond from the side-

chain amide N–H of Gln²⁸. The glycerol carbon chain

is in hydrophobic contact with Leu²⁰.²⁸In contrast, in

the structures of human DHFR complexes containing

dihydrofolate or 2, this site is well packed with hydro-

phobic side-chains.^32,33Since this glycerol is clearly

tightly and speciﬁcally bound in a ﬁxed conformation

close to N(8) of 2, we designed series of molecules which

contain a 1,2,3-triol joined to a head group which would

mimic the binding of 2 deep in the dihydrofolate-binding

pocket.

Since 3 is a weak inhibitor of M. avium DHFR activity³⁴

and many other pyrimidine-2,4-diamines inhibit various

DHFRs, we chose pyrimidine-2,4-diamine as the tem-

plate to which to attach the linker from the triol. Com-

pounds 7 (Scheme 1) were designed directly from

modelling the orientation of the glycerol and overlay

of the pyrimidine-2,4-diamine unit with the diaminop-

teridine of 2. This overlay suggested that a two-carbon

linker (-CH₂CH₂-) would be optimum to join the triol

to the pyrimidine 6-position; it also showed the need

for R conﬁguration at the C(3) secondary alcohol of

R⁴

R³

R⁴

R³

R⁴

NH₂

N

NH₂

N

NH₂

R³

N

R³

N

R⁴

N

OH

H₂N

N

H₂N

N

OH

10

OH

9

7

8

OH

R⁴

R³

R⁴

R³

R⁴

R³

NH₂

NC

MeO

NC

N

EtO₂CR

15

R

O

R

H₂N

N

R

11: R = (CH₂)₂Ph

12: R = Me

13

14

16

R = 3R,4S-3,4,5-trihydroxypentyl, 3S,4S-3,4,5-trihydroxypentyl, 5-hydroxypentyl, 1S,2R-1,2,3-

trihydroxypropyl, Ph(CH₂)₂, Me

a: R³= R⁴= H; b: R³= H, R⁴= Cl; c: R³= H, R⁴= Br; d: R³= R⁴= Cl.

Scheme 1. Structures of designed pyrimidine-2,4-diamines 7–12 and retrosynthetic analysis.

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4555

the 3,4,5-trihydroxypentyl side-chain (mimicking glyc-

erol O(1)) and S conﬁguration at the C(4) secondary

alcohol (mimicking glycerol O(2)), as in 7. The diaste-

reomeric series 8 is S at C(3); this series tests the validity

of the drug design, since the linker length is the same as

in 7 but the orientation of the triol relative to the pyrim-

idine-diamine should not be apposite for binding. In 9,

the secondary alcohols are missing, leaving only the

primary alcohol of the 6-(5-hydroxypentyl) group to

mimic O(3) of the glycerol and H bond to Leu²⁴in the

glycerol-binding pocket, losing the ability to H bond

to Trp²², Asp²⁷and Gln²⁸, but retaining possible hydro-

phobic interactions with Leu²⁰. The length of the linker

between the triol and the pyrimidine-2,4-diamine is

tested in the 6-(1,2,3-trihydroxypropyl) compounds 10;

these compounds retain the triol motif with the same

conﬁguration at the secondary alcohols as in 7 but

joined directly to pyrimidine C(6).

guanidine with enol ethers derived from a-acylphenyl-

acetonitriles, used by Russell and Hitchings¹⁶in their

syntheses of pyrimethamine 3, etoprine 4 and related

antimalarial compounds carrying simple small-alkyl

substituents at the 6-position of the pyrimidine-2,4-di-

amine core. Tarnchompoo et al.¹⁹have extended this

synthetic approach to analogues carrying larger alkyl

and x-arylalkyl groups at this position, in their search

for pyrimidine-2,4-diamines which inhibit DHFR

activity in P. falciparum which is resistant to 3. The

acylation steps and the protection of the OH groups

were optimised individually for each series of target

compounds.

3.2. Synthesis of 5-aryl-6-((3R,4S)-3,4,5-trihydroxy-

pentyl)pyrimidine-2,4-diamines (7)

Scheme 2 shows our approach to the 5-aryl-6-((3R,4S)-

3,4,5-trihydroxypentyl)pyrimidine-2,4-diamines 7, using

protection for the primary alcohol. We rationalised

that the ester 21 would provide the required masked

triol at the 6-position and could be synthesised by a

two-carbon chain extension from a protected ^L-ery-

throse 18. Acetonide protection was introduced be-

tween the cis 3-OH and 4-OH of ^L-arabinose 17 by

acid-catalysed reaction with 2,2-dimethoxypropane.

Oxidative cleavage of the C(1)–C(2) bond with perio-

date then gave ^L-erythrose-2,3-acetonide 18. The re-

quired two-carbon chain-extension was achieved by

base-free Wittig reaction of the latent aldehyde of 18

with pre-formed ethyl triphenylphosphoranylidineace-

tate to aﬀord the stereoisomeric a,b-unsaturated esters

19E and 19Z in 69% overall yield (ratio of geometrical

isomers 3:11, 19E and 19Z, respectively). These geo-

metrical isomers were readily separated chromato-

In each of the sets of 6-((poly)hydroxyalkyl)pyrimi-

dine-2,4-diamines 7–10, a phenyl is located at posi-

tion-5 of the pyrimidine, to occupy

a (largely)

hydrophobic pocket which the hinge region

(-CH₂NMe-) of 2 occupies in Figure 2a. This phenyl

is unsubstituted in 7a–10a, whereas this ring is haloge-

nated in other designed compounds. It carries a 4⁰-

chlorine in 7b–10b (reﬂecting the 4⁰-chlorine in 3)

and a 4⁰-bromine in 7c–10c. 3⁰,4⁰-Dichlorophenyl was

incorporated into 7d, 9d and 10d to mimic the dichlor-

ophenyl in 4a and 4b; the corresponding analogue in

the 8 series was planned but was synthetically inacces-

sible. Compounds 11 and 12 (Scheme 1) were designed

as gross tests of the structure-based design of inhibi-

tors, while retaining the essential pyrimidine-2,4-dia-

mine. In 11, the designed triol is replaced by a

hydrophobic aromatic benzene ring which should

interact unfavourably with the H bonding environ-

ment of the glycerol-binding pocket. In 12, there is

no group which may enter this pocket.

1

graphically and were identiﬁed on the basis of the H

NMR coupling constants in the –HC@CH– system.

Separation of the isomers was unnecessary in the syn-

thetic plan, as catalytic hydrogenation of the mixture

of 19E and 19Z gave the saturated ester 20 quantita-

1

tively. H NMR spectroscopy conﬁrmed the presence

3. Chemical synthesis

3.1. Synthetic strategy

of only one diastereoisomer of 20. A variety of protect-

ing groups was investigated for the primary alcohol; we

proposed that this alcohol should not be exposed dur-

ing the reaction of the ester with the carbanion derived

from the phenylacetonitriles, to avoid possible quench-

ing of the carbanion and to avoid lactonisation of the

hydroxy-ester 20. The primary alcohol of 20 was ben-

zylated by generation of the alkoxide with lithium

bis(trimethylsilyl)amide and reaction with benzyl bro-

mide to give the fully protected ester 21. The classical

conditions for using esters to acylate phenylacetonitrile

carbanions,¹⁶sodium ethoxide in ethanol, failed to ef-

fect the required reaction. However, the carbanions

were generated from the (halo)phenylacetonitriles un-

der aprotic conditions with lithium bis(trimethyl-

silyl)amide in diethyl ether at low temperature; these

reacted with 21 to aﬀord the a-acylphenylacetonitriles

22a–d in 16–26% yields. The ¹H NMR spectra indi-

cated the presence of varying amounts of the enol

tautomers 23a–d. Methylation with diazomethane gave

the enol ethers 24a–d as inseparable mixtures of

geometrical isomers. Condensation of these mixtures

The planned synthetic approaches to the series of target

pyrimidine-2,4-diamines 7–12 are shown in retrosynthet-

ic format in Scheme 1. In each case, condensation of an

appropriately substituted corresponding enol ether 13

with guanidine would furnish the pyrimidinediamine.

The enol ethers would be readily prepared by methyla-

tion of the a-acylphenylacetonitriles 14, which in turn

would be available by acylation of anions derived from

(Ar-substituted) phenylacetonitriles 15 with the appro-

priate esters 16, with or without protection of the side-

chain alcohols. Several questions needed to be addressed

during the development of the synthetic routes: how

should the condensation with guanidine be optimised?

How should the acylation be optimised? Do the primary

and secondary alcohols in the side-chains need to be

protected during the acylation or condensation steps?

If so, what should the protecting groups be? We elected

to use the general synthetic approach, condensation of

4556

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

EtO₂C

CO₂Et

OH

OR

O

HO

O

i,ii

iii

iv

+

HO

O

HO

O

OH

vi

17

18

19E

19Z

20: R = H

21: R = Bn

v

vi

R³

R⁴

R³

NC

NH₂

R⁴

N

R³

N

RO

viii

O

ix

OBn

H₂N

N

H₂N

N

OBn

OH

O

26

OH

25

O

x

xi

23: R = H

22

vii

24: R = Me

R³

R⁴

R³

R⁴

NC

NH₂

R⁴

N

R³

N

ix

viii

RO

O

OH

H₂N

N

H₂N

N

OH

O

7

OH

30

O

a: R³= R⁴= H; b: R³= H, R⁴= Cl;

c: R³= H, R⁴= Br; d: R³= R⁴= Cl

28: R = H

29: R = Me

27

vii

Scheme 2. Synthetic routes to 5-aryl-6-((3R,4S)-3,4,5-trihydroxypentyl)pyrimidine-2,4-diamines 7, using Bn protection for the primary OH and

omitting protection for the primary OH. Reagents: (i) Me₂C(OMe)₂, TsOH, DMF; (ii) NaIO₄, H₂O, hexane; (iii) EtO₂CCH@PPh₃, CH₂Cl₂; (iv) H₂,

Pd/C, EtOH; (v) LiN(SiMe₃)₂, BnBr, THF, DMF; (vi) LiN(SiMe₃)₂, ArCH₂CN, Et₂O; (vii) CH₂N₂, Et₂O; (viii) guanidineÆHCl, NaOMe,

MeO(CH₂)₂OH; (ix) aq CF₃CO₂H; (x) Na, liquid NH₃; (xi) FeCl₃, CH₂Cl₂.

with guanidine in boiling 2-methoxyethanol then led to

the pyrimidine-2,4-diamines 25a–d in satisfactory

yields; similar reactions in the conventional solvent

for these condensations, ethanol, gave lower yields.

ration of the halogenated congeners 7b–d, as reduction

of the carbon–halogen bonds led to exclusive formation

of the phenyl analogue 7a from 26b–d. The most gener-

ally applicable debenzylation for this series was the use

of the Lewis acid anhydrous iron(III) chloride in dichlo-

romethane, as developed by Park et al.³⁶By this meth-

od, 26b–d were converted in high yields into the

required triols 7b–d. Moreover, the Lewis acidity of this

reagent could be exploited also in removal of the aceto-

nides, in that both acetonide and benzyl ether protecting

groups could be removed from 25a–d in one pot to fur-

nish 7a–d directly, albeit in lower overall yields than in

the two-step processes. TLC analysis suggested that, in

this one-pot process, the acetonide was cleaved within

5 min and the debenzylation was essentially complete

within 80 min.

Removal of the acetonide protection from 25a–d with aq

triﬂuoroacetic acid revealed the secondary alcohols in

26a–d in excellent yields but subsequent removal of the

benzyl protection from the primary alcohol was more

challenging. Catalytic hydrogenolysis (H₂, Pd/C, various

solvents) failed to remove the benzyl group from 26a,

even in the presence of catalytic perchloric acid. How-

ever, addition of a catalytic amount of chloroform³⁵to

the hydrogenolysis reaction mixture in methanol facili-

tated the deprotection to give triol 7a. This method

could not be extended to debenzylation of the halo-

gen-bearing analogues 26b–d, as hydrogenolysis of the

carbon–halogen bonds occurred; 26b and 26c gave 7a

only, whereas 26d gave an inseparable mixture of 7a,

7b and the meta-monochloro analogue. Attempted deb-

enzylation with hydrogen bromide in acetic acid, an-

other common method, gave regioisomeric mixtures

of bromo- and acetoxy-pentylpyrimidine-2,4-diamines.

The most eﬀective method for preparation of the Ar-

unsubstituted analogue 7a was reductive cleavage of

the O-benzyl protecting group with sodium in liquid

ammonia. This method could not be extended to prepa-

In view of these challenges, the assembly of the pyrimi-

dine ring was attempted with a free primary alcohol in

the side-chain. As shown in Scheme 1, the phenylaceto-

nitriles were deprotonated with lithium bis(trimethylsi-

lyl)amide and the anions were quenched with the ester

20. Use of two equivalents of base was necessary to

achieve condensation to obtain the a-acylphenylaceto-

nitriles 27 in a maximum yield of 10%, indicating that

protection of the primary alcohol is beneﬁcial for this

acylation to proceed eﬃciently. Methylation of the

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4557

tautomeric enols 28 with diazomethane and condensa-

tion of the enol ethers 29 with guanidine gave the pyrim-

idine-2,4-diamines 30. Again, the yields were

signiﬁcantly lower with the exposed primary alcohol

(30a, 42%; 30b, 12%; 30c, 20%; 30d, 9%). Deprotection

was straightforward to furnish the target triols 7.

was immediately condensed with ethyl triphenylphos-

phoranylidineacetate in a Wittig reaction to give the

chain-extended a,b-unsaturated esters 37E and 37Z.

In contrast to the analogous uncatalysed formation

of 19E and 19Z (which carry free primary alcohols)

at ambient temperature, this reaction required pro-

longed heating at 110 ꢁC and catalysis with benzoic

acid. In this case, the mixture of the separable geomet-

rical isomers 37E and 37Z was approximately equimo-

lar. Careful control of the hydrogenation conditions

was required to reduce the alkene of the 37E/37Z mix-

ture to form key intermediate 38 without causing loss

of the benzyl protecting group through hydrogenolysis.

The fully protected ester 38 was then used, as for the

diastereomer 21, to alkylate the carbanions derived

from the (halo)phenylacetonitriles to aﬀord the a-acyl-

phenylacetonitriles 39a–c; 3,4-dichlorophenylacetonitri-

le failed to react. Methylation of the enols 40 and

condensation of the enol ethers 41 with guanidine led

to the pyrimidine-2,4-diamines 42, in much higher

yields (42a, 67%; 42b, 48%; 42c, 53%) than in the R,

S series. The side-chain alcohols were deprotected in

two steps. Acid-hydrolysis of the acetonide rapidly

gave the diols 43. As in the diastereomeric series,

hydrogenolysis removed the benzyl group from 43a

to aﬀord 8a in high yield; debenzylation with iron(III)

chloride converted 43b and 43c to the triols 8b and 8c,

respectively, avoiding the dehalogenations associated

with other debenzylation procedures.

3.3. Synthesis of 5-aryl-6-((3S,4S)-3,4,5-trihydroxypen-

tyl)pyrimidine-2,4-diamines (8)

The approach to the diastereomeric 5-aryl-6-((3S,4S)-

3,4,5-trihydroxypentyl)pyrimidine-2,4-diamines 8 was

broadly similar to that for 7, using the benzyl protec-

tion method. In this series (Scheme 3), the key interme-

diate was the trans-dioxolane 38, a diastereomer of the

cis-dioxolane ester 21 above. The approach to 38

started with protection of the secondary alcohols of

diethyl R,R-tartrate 31 as the acetonide 32; these sec-

ondary alcohols will become the secondary alcohols

of the targets 8 with the appropriate conﬁgurations.

Reduction with lithium aluminium hydride furnished

the C₂-symmetric diol 33. Mono-protection of this diol

was essential for developing the chain-extension of only

one arm. The optimum conditions were found to be

deprotonation with one equivalent of sodium hydride

in DMF, followed by alkylation with benzyl chloride,

giving the required monoether 35, with a trace of

diether 34. Pyridinium chlorochromate oxidation con-

verted the exposed alcohol to the aldehyde 36, which

H H

EtO₂C

CO₂Et

HO

OH BnO

iii

OBn

EtO₂C

HO

CO₂Et

i

OH

O

31

32

33

34

EtO₂C

+

OHC

OBn

OBn HO

iv

OBn

vi

v

O

38

37Z/

E

36

35

vii

R⁴

NC

HO

NC

NH₂

N

R⁴

N

O

MeO

OBn

H₂N

OBn

ix

viii

O

39

40

41

42

x

R⁴

NH₂

xi

xii

N

OH

OBn

OH

H₂N

N

H₂N

N

a: R⁴= H

b: R⁴= Cl

c: R⁴= Br

8

OH

43

OH

˚

Scheme 3. Synthesis of 5-aryl-6-((3S,4S)-3,4,5-trihydroxypentyl)pyrimidine-2,4-diamines 8. Reagents and condition: (i) Me₂C(OMe)₂, TsOH, 4 A

˚

molecular sieve, CH₂Cl₂; (ii) LiAlH₄, THF; (iii) NaH, BnCl, DMF; (iv) PCC, NaOAc, 4 A molecular sieve, CH₂Cl₂; (v) EtO₂CCH = PPh₃, PhCO₂H,

PhMe, D; (vi) H₂, Pd/C, EtOH; (vii) LiN(SiMe₃)₂, ArCH₂CN, Et₂O; (viii) CH₂N₂, Et₂O; (ix) guanidineÆHCl, NaOMe, MeO(CH₂)₂OH; (x) aq

CF₃CO₂H; (xi) H₂, Pd/C, EtOH; (xii) FeCl₃, CH₂Cl₂.

4558

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

R⁴

R³

3.4. Synthesis of 5-aryl-6-(5-hydroxypentyl)pyrimidine-

2,4-diamines (9)

NC

O

Although a similar approach of protection of the pri-

mary alcohol could have been used in the syntheses

of 6-(5-hydroxypentyl)pyrimidine-2,4-diamines 9,

O

i

O

a

O

strategy was devised to use a lactone to provide the

necessary acylating ester, simultaneously masking the

primary alcohol (Scheme 4). The carbanions of the

(halo)phenylacetonitriles were generated in the usual

way with lithium bis(trimethylsilyl)amide; the yields

of the reactions with lactone 44 to give the a-(6-

hydroxyhexanoyl)phenylacetonitriles 45 were low but

provided suﬃcient material for further methylation of

the enols 46 and condensation of 47 with guanidine

to give the required 6-(5-hydroxypentyl)pyrimidines 9

in moderate yields. No deprotection steps were re-

quired in this series as the primary alcohols had been

revealed during the reaction of the lactone with the

phenylacetonitrile anions.

OH

48

49

R⁴

R³

NH₂

N

NC

RO

N

R³

iii

O

H₂N

O

OH

50: R = H

51: R = Me

52

iv

ii

R⁴

R³

NH₂

N

OH

a: R³= R⁴= H

H₂N

b: R³= H, R⁴= Cl

c: R³= H, R⁴= Br

d: R³= R⁴= Cl

3.5. Synthesis of 5-aryl-6-((1S,2R)-1,2,3-trihydroxypro-

pyl)pyrimidine-2,4-diamines (10)

10

OH

The lactone strategy was also used for the chain-short-

ened triols 10 (Scheme 5). 2,3-O-Isopropylidene-^D-ery-

thronolactone 48 reacted with the phenylacetonitrile

anions to aﬀord 49 in 21–38% yields. In the usual way,

methylation of the enols 50, condensations of the enol

ethers 51 with guanidine and aqueous acid deprotection

of 52 gave the pyrimidine-2,4-diamines 10 carrying the

6-((1S,2R)-1,2,3-trihydroxypropyl) side-chains.

Scheme 5. Synthesis of 5-aryl-6-((1S,2R)-1,2,3-trihydroxypropyl)

pyrimidine-2,4-diamines 10. Reagents: (i) LiN(SiMe₃)₂, ArCH₂CN,

Et₂O; (ii) CH₂N₂, Et₂O; (iii) guanidineÆHCl, NaOMe, MeO(CH₂)₂OH;

(iv) aq CF₃CO₂H.

two chiral centres. Thus the Ph 2-H and 6-H become

diastereotopic, as do the Ph 3-H and 5-H. For example,

in the H NMR spectrum of 52a, the Ph 2-H signal is

The dioxolanylpyrimidine intermediates 52 carry two

bulky groups in close proximity in the 5- and 6-positions

of the pyrimidine. MM2 energy minimisation suggests

that this twists the 5-(4-halo)phenyl group in 52a–c

out of the pyrimidine plane by ca. 60ꢁ (Fig. 3). The re-

stricted rotation about the pyrimidine–Ph bond is evi-

dent in the NMR spectra of these compounds. The

benzene ring is held close to the dioxolane, which bears

1

separated from the Ph 6-H signal by 0.21 ppm, whereas

the 3-H and 5-H signals are coincident. In the spectrum

of the 4-chloro compound 52b, the Ph 2-H and 6-H sig-

nals are separated by 0.22 ppm and the 3-H and 5-H

signals are separated by 0.04 ppm. In the spectrum of

the 4-bromo compound 52c, the separations are

0.05 ppm and 0.02 ppm, respectively. In 52a–c, the sub-

stituents, if present, are in the 4-position of the benzene

ring and are therefore coaxial with the pyrimidine–ben-

zene bond. However, 52d carries a chlorine atom in po-

sition-3 of the benzene ring, which is oﬀ the axis of this

bond. Therefore, two diﬀerent conformers 52dA and

52dB can exist, as shown in Figure 3 in stick and

space-ﬁlling representations. Conformers 52dA and

52dB are diastereoisomers of very similar energy,

according to MM2 calculations. The ¹H NMR spectrum

of 52d shows the presence of both conformers in 1:1 ra-

tio; the sharpness of the signals indicates that, as could

be predicted from the severe steric crowding, intercon-

version is slow. The ¹H signals for 2-H for the diastereo-

meric conformers are separated by 0.10 ppm, the signals

for 5-H by 0.02 ppm and the signals for 6-H by

R⁴

O

NC

R³

O

i

O

(CH₂)₅OH

44

45

R⁴

R³

R⁴

iii

NH₂

N

NC

RO

N

R³

H₂N

(CH₂)₅OH

9

46: R = H

47: R = Me

ii

1

0.10 ppm. Other H NMR signals are co-incident for

a: R³= R⁴= H; b: R³= H, R⁴= Cl

c: R³= H, R⁴= Br; d: R³= R⁴= Cl

the two conformers, as are all the peaks in the ¹³C

NMR spectrum. The latter was assigned by analogy

with the spectra for 3 and related compounds examined

in detail earlier.³⁷This eﬀect was not observed for the

triols 10a–d and only one set of signals could be seen

Scheme 4. Synthesis of 5-aryl-6-(5-hydroxypentyl)pyrimidine-2,4-

diamines 9. Reagents: (i) LiN(SiMe₃)₂, ArCH₂CN, Et₂O; (ii) CH₂N₂,

Et₂O; (iii) guanidineÆHCl, NaOMe, MeO(CH₂)₂OH.

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4559

R⁴

NC

O

EtO₂C

i

R

54: R = Bn

58: R = H

62: R = Me

55: R = Bn; R⁴= H

59: R = H; R⁴= H

63: R = Me; R⁴= Cl

R⁴

NH₂

NC

N

iii

H₂N

N

R'O

R

11: R = Bn; R⁴= H 56: R = Bn; R' = R⁴= H

12: R = H; R⁴= H 57: R = Bn; R' = Me, R⁴= H

3: R = Me; R⁴= Cl 60: R = R' = R⁴= H

61: R = R⁴= H; R' = Me

ii

Figure 3. MM2-minimised structures of pyrimidine-2,4-diamines 52a–

d, showing the steric interactions between the 5-(halo)phenyl group

and the 6-(2,2-dimethyl-5-hydroxymethyl-1,3-dioxolan-4-yl) substitu-

ent. As a result of this steric crowding, the (halo)phenyl group is

twisted to ca. 60ꢁ from the plane of the pyrimidine. Compound 52d

exists as two diastereomeric conformers, which are evident in the ¹H

and ¹³C NMR spectra.

64: R = Me; R' = H, R⁴= Cl

65: R = R' =Me, R⁴= Cl

Scheme 6. Synthesis of 5-phenyl-6-(2-phenylethyl)pyrimidine-2,4-dia-

mine 11, 6-methyl-5-phenylpyrimidine-2,4-diamine 12 and pyrimeth-

amine 3. Reagents: (i) LiN(SiMe₃)₂, ArCH₂CN, Et₂O; (ii) CH₂N₂,

Et₂O; (iii) guanidineÆHCl, NaOMe, MeO(CH₂)₂OH.

for each compound, with 2-H and 6-H being magneti-

cally equivalent. This probably reﬂects the greater

ﬂexibility in the triol side-chain. The eﬀects were also

not observed for the homologues 7 and 8, also owing

to increased ﬂexibility and the remoteness of the chiral

dioxolane from the benzene ring in these structures

(Fig. 4).

starting with generation of the anion from 4-chloro-

acetonitrile with LiN(SiMe₃)₂and reaction with ethyl

propanoate 61 to give 62. Enol 63 was methylated, giv-

ing 64; condensation with guanidine in hot 2-methoxy-

ethanol provided 3 in good yield.

3.6. Synthesis of pyrimidine-2,4-diamines 11, 12 and 3,

lacking OH in the 6-substituent

4. Biological evaluation

4.1. Inhibition of DHFR activities

Three pyrimidine-2,4-diamines 11, 12 and 3, lacking

alcohols in the 6-substituent, were required as controls

in the biological evaluation. The synthetic approaches

followed the general sequence (Scheme 6). Acylation

of phenylacetonitrile anion with ethyl 2-phenylpropano-

ate 53, methylation of 55 and condensation of 56 with

guanidine gave 6-(2-phenylethyl)pyrimidine-2,4-diamine

11. The minimal analogue 12 was prepared similarly,

through acylation of phenylacetonitrile anion with ethyl

acetate 57, methylation of 59 and condensation of 60

with guanidine. Finally, 3 was produced in a new route

Direct screening of candidate drugs with M. tuberculosis

is slow and requires biosafety Level 3 facilities and pro-

cedures.³⁸The slow growth of M. tuberculosis has been

frustrating, with most public health laboratories still

employing cultivation techniques that require 3–6 weeks

to achieve growth. This mainly reﬂects the slow genera-

tion time inherent in the organism. Mycobacterium

smegmatis and M. avium have often been used as surro-

gates for assessment of activity of candidate drugs, as

they grow rapidly and are less pathogenic to hu-

mans.^39–41However, drug screening in wild-type M.

smegmatis has not always been an accurate predictor

of activity⁴²or of mechanism of action in M.

tuberculosis.⁴³

A new approach to screening compounds for selective

inhibition of DHFR from M. tuberculosis has been

developed by Gerum et al.³⁸In this, the TH5 strain of

the yeast Saccharomyces cerevisiae, which lacks endoge-

nous expression of DHFR, was engineered to contain a

vector p414CYC1 carrying a single copy of the dfrA

gene from M. tuberculosis. This gene codes for the pro-

tein with DHFR activity in M. tuberculosis. The native

TH5 strain of S. cerevisiae requires supplementation

Figure 4. Typical plate (DMSO only control) showing orthogonal

streaks of yeast after 3 days of incubation.

4560

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

with dTMP, uracil, adenine and a full complement of

amino acids to grow, whereas the engineered strain con-

taining the dfrA gene can grow normally. Thus inhibi-

tion of the expressed M. tuberculosis DHFR activity

would be manifested as inhibition of growth of the

yeast. Two engineered TH5-derived strains of S. cerevi-

siae were also engineered to carry yeast or human

DHFR genes. Inhibition of the growth of these yeasts

by test compounds would indicate that these eukaryotic

DHFRs are inhibited and would point to lack of selec-

tivity for the prokaryotic M. tuberculosis enzyme. These

three engineered yeasts were kindly supplied by Dr.

Carol Hopkins Sibley (Department of Genome Sciences,

University of Washington, Seattle, Washington, USA).

Thus, in the present work, the test compounds were

evaluated for their ability to inhibit selectively the

growth of yeast carrying M. tuberculosis DHFR, while

having less inhibition of yeast bearing either the yeast

or the human enzyme. This assay, performed on a spoke

assay plate, is semi-quantitative; comparison of the

diameters of the zones of inhibition of the three yeasts

by a particular test compound gives an indication of

the selectivity of inhibition of the M. tuberculosis DHFR

by that compound. Compounds can also be ranked

approximately for potency of inhibition, although no

quantitative IC₅₀data can be derived.

Table 1 shows the mean diameters of the zones of inhibi-

tion of growth of the three yeasts by the pyrimidine-

2,4-diamines 7–10 carrying one or more alcohols in the

sidechain, by the pyrimidine-2,4-diamines 11 and 12 with

simple lipophilic side-chains and by the known DHFR-

inhibiting pyrimidine-2,4-diamines 3 and 6. Data for

the negative control, DMSO without drug, are also gi-

ven. Trimethoprim 6 has been reported to have a broad

spectrum of activity against gram-positive bacteria,

including methicillin-sensitive (MSSA) and methicillin-

resistant (MRSA) S. aureus, and gram-negative bacteria,

including E. coli, but less activity or no activity against

Mycobacterium spp., Pseudomonas aeruginosa and Chla-

mydia pneumoniae.⁴⁴At the enzymic level, 6 is only a

weak inhibitor of M. avium DHFR and of eukaryotic

DHFR but is potent in inhibiting DHFR activity in sus-

ceptible bacteria.⁴¹In line with these reports, 6 was

found to be inactive against all three DHFRs in this yeast

assay. Pyrimethamine 3 was very poorly active, even

against the human enzyme, despite being reported to

have K_i= 58 nM against human DHFR.¹⁷This observa-

Table 1. Diameters of zones of inhibition of growth of S. cerevisiae carrying the DHFR gene from M. tuberculosis, S. cerevisiae carrying the human

DHFR gene and wild-type S. cerevisiae by test pyrimidine-2,4-diamines 7–10 and by control pyrimidine-2,4-diamines 11, 12, 3 (pyrimethamine) and 6

(trimethoprim)

R⁴'

NH₂

N

R³'

H₂N

N

R⁶

0

Compound

R³

R⁴

R⁶

Diameter of zone of

inhibition (mm)

S. cerevisiae

(TB-DHFR)^a,b

Diameter of zone

of inhibition (mm)

S. cerevisiae

(human-DHFR)^a,c

Diameter of zone

of inhibition (mm)

S. cerevisiae

(yeast-DHFR)^a,d

7a

7b

H

Cl

H

Cl

H

Cl

H

Cl

H

(3R,4S)-3,4,5-trihydroxypentyl

(3S,4S)-3,4,5-trihydroxypentyl

5-Hydroxypentyl

11

9

7

8

9

7

8

7

8

9

5

8

5

7

5

6

7

6

8

7

5

7

8

9

5

8

5

8

6

7

8

6

9

8

9

5

7

5

8

5

6

5

Cl

Br

Cl

H

7c

7d

8a

8b

Cl

Br

H

8c

9a

9b

9c

Cl

Br

Cl

H

5-Hydroxypentyl

9d

10a

(1S,2R)-1,2,3-trihydroxypropyl

2-Phenylethyl

10b

10c

Cl

Br

Cl

H

10d

20

12

H

Me

Ethyl

3 (pyrimethamine)

6 (trimethoprim)

DMSO negative control

H

^aDiameters of the zone of inhibition were measured for each of the orthogonal streaks on each of at least three test plates for each determination;

data are expressed 1 mm.

^bTB5 yeast engineered to contain DHFR from M. tuberculosis only.

^cTB5 yeast engineered to contain human DHFR only.

^dTB5 yeast engineered to contain yeast DHFR only.

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4561

tion suggests that, despite being only semi-quantitative,

the assay is a stringent test of inhibitory activity. The

pyrimidine-2,4-diamine 11, which lacks hydroxy groups

and carries only lipophilic substituents, was also inactive

against all the DHFRs. Interestingly, the minimal lipo-

philic pyrimidine-2,4-diamine 12, which bears only a

methyl group at position-6, showed some inhibitory

activity, although it was unselective.

essary to use the binding contacts of one primary and

the secondary alcohol of the glycerol, in that the 6-(5-

hydroxypentyl) compounds 9 are not selective for the

M. tuberculosis enzyme. Third, the length of the linker

joining the dihydrofolate mimic (the pyrimidine-2,4-dia-

mine) to the glycerol mimic is critical; shortening the

distance in 10 abolishes activity.

4.2. Modelling of the selective inhibitor 7a in the dihydro-

folate- and glycerol-binding sites of M. tuberculosis DHFR

Pyrimidine-2,4-diamines 7a–d, which carry the (3R,4S)-

3,4,5-trihydroxypentyl side-chain at the 6-position, were

designed to mimic directly the pteridine of the dihydro-

folate and the glycerol, with the conﬁguration of each

chiral centre being as predicted by the structure-based

design; the –CH₂CH₂– linker is also of the length indi-

cated by the modelling studies to be apposite. Within

this set, the 5-phenyl compound 7a showed notable

selectivity for inhibition of the growth of the yeast con-

taining the M. tuberculosis DHFR, with only very mod-

est inhibition of the growth of the yeasts containing the

Homo sapiens enzyme or the S. cerevisiae enzyme. The

4⁰-chlorophenyl analogue 7b also showed some selectiv-

ity for inhibition of the M. tuberculosis enzyme, whereas

the 4⁰-bromophenyl and 3⁰,4⁰-dichlorophenyl com-

pounds 7c and 7d had modest and equivalent activity

against each DHFR.

The structures of selected pyrimidines from the series

were modelled into the dihydrofolate-binding site and

the glycerol pocket, to attempt to rationalise the struc-

ture–activity observations and thus to validate the de-

sign process. The compounds were bound into the

dihydrofolate and glycerol binding pockets using the

H-bonds from the pyrimidine-2,4-diamine ring to estab-

lish an orientation similar to the observed binding con-

formation of methotrexate 2.²⁸The triol section was

then docked using the H-bonds established from the

bound glycerol in the X-ray structure (as distance re-

straints). Molecular dynamics calculations were then

performed on the bound ligand using the H-bonds (X-

ray observed) as distance restraints between the bound

ligand and the pocket. The ligand was ramped to

300 K over a period of 10 ps and then held at 300 K

for 20 ps. Observing the conformations over the ﬁnal

20 ps gave two distinct binding conformers. Throughout

the above procedure, the binding pocket was restrained

and only the ligand was allowed to change orientation.

Average structures were taken (7–13 and 15–20 ps)

which were then minimised within a restrained binding

pocket. The two structures obtained were then freely

minimised (ligand and binding pocket to a radius of

The diastereomeric series 8a–c showed modest activity

but little evidence of selectivity. Removal of the second-

ary alcohols from the 6-position side-chain, in 9, led to

compounds with increased potency but completely lack-

ing selectivity. In contrast, shortening the side-chain by

removal of the –CH₂CH₂– linker but retaining the con-

ﬁguration of the secondary alcohols eﬀectively abolished

inhibitory activity in 9a–c but the 3⁰,4⁰-dichlorophenyl

compound 9d showed modest but non-selective inhibi-

tion of all the DHFRs.

˚

15 A) to give the structures and conformations shown

in Figure 5.

Several trends are noticeable in the structure–activity

relationships for these pyrimidine-2,4-diamines. First,

comparison of the results for 7a and b with those for

the diastereoisomers 8a and b indicates that the conﬁgu-

ration of the hydroxy groups is critical for selective inhi-

bition of M. tuberculosis DHFR, as predicted by the

model. Second, the secondary alcohols appear to be nec-

Figure 5 shows the occupation of these sites by the two

conformers of 7a, the most selective inhibitor of M.

tuberculosis DHFR. As expected, the triol makes H-

bonds with Asp²⁷, Gln²⁸, Leu²⁴and Trp²², following

the pattern shown by the glycerol in the crystal struc-

ture.²⁸With the glycerol-mimicking triol held by the

hydrogen-bonding network, the pyrimidine-2,4-diamine

Figure 5. Images of structures of DHFR from M. tuberculosis, with 7a bound at the dihydrofolate-binding site (structure derived from modelling

study, see text). (a) View of the structure of M. tuberculosis DHFR with 7a bound in conformation 7aA; (b) View of the structure of M. tuberculosis

DHFR with 7a bound in conformation 7aB.

4562

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

is perfectly located for its own hydrogen-bonding inter-

actions deep in the dihydrofolate-binding site. These

constraints place the 5-phenyl substituent of 7a in a

pocket of limited size. Indeed, this pocket cannot

accommodate halogens in the 4⁰-position of the phenyl,

as this position is tight against the surface of the enzyme;

thus the observations that the 4⁰-bromo and 3⁰,4⁰-

dichloro analogues (7c and 7d, respectively) are not

selective inhibitors are rationalised in the model. The

4⁰-chloro analogue 7b, however, does show slight selec-

tive inhibition of M. tuberculosis DHFR and it may be

possible to accommodate the chlorine, albeit with a sig-

niﬁcant penalty in displacing the other binding contacts

from their ideal positions.

mycobacterial DHFR. Compounds 10 tested the length

of the linker between the triol moiety and the pyrimi-

dine-2,4-diamine.

The target compounds were synthesised by acylation of

the anions derived from phenylacetonitriles with appro-

priately functionalised and protected esters and lac-

tones, followed by methylation, condensation with

guanidine and deprotection, if appropriate. The acyla-

tion step was optimised as generation of the phenyl-

acetonitrile anion with lithium bis(trimethylsilyl)amide

at ꢀ78 ꢁC, followed by addition of the ester or lactone.

Yields under these optimised conditions ranged from 6%

to 41%, with the lower yields being obtained with sub-

strates containing unprotected alcohols. The condensa-

tions with guanidine were generally uneventful and

high yielding. Removal of benzyl groups presented a

particular challenge, as many reductive methods also

eﬀected dehalogenation in some analogues.

The active lead compound 7a can adopt two diﬀerent con-

formations. As with all 5-(substituted)phenyl-6-substi-

tuted-pyrimidine-2,4-diamines, the 5-phenyl ring of 7a

has to be twisted out of the plane of the aromatic hetero-

cycle to accommodate the adverse steric interactions

between the phenyl ortho-hydrogens and the adjacent

4-NH₂and 6-substituent. This rotation about the Ph–

pyrimidine bond can be either clockwise or anticlockwise

to achieve the same relief of steric strain. In conformer

7aA, the phenyl is rotated anticlockwise from coplanarity,

whereas clockwise rotation produces 7aB; these conform-

ers are almost identical in energy in free space. However,

7aA ﬁts well into the pocket in the M. tuberculosis DHFR

(Fig. 5a), whereas the forward edge of the 5-phenyl of 7aB

is located tightly pressed against the top of the enzyme

pocket (Fig. 5b). Thus the calculated energy of the com-

plex of M. tuberculosis DHFR with conformer 7aA is of

consistently higher energy than that of the complex of

M. tuberculosis DHFR with conformer 7aB; indicating

that 7a binds in conformer 7aA.

Evaluation of the test 6-substituted pyrimidine-2,4-

diamines for their inhibition of the growth of yeasts

containing active DHFR from human, M. tuberculosis

and yeast indicated that one compound, 7a, was selec-

tive for inhibition of M. tuberculosis DHFR and did

not inhibit human DHFR or yeast DHFR signiﬁcantly

in the assay. Other compounds were inactive or less ac-

tive. Modelling the structure of 7a into the dihydrofo-

late- and glycerol-binding pockets of M. tuberculosis

DHFR rationalised the inhibition data, validating the

original design of selective inhibitors and explaining

the negative eﬀect of halogenation of the 5-phenyl ring

on biological activity. These modelling studies also

indicated which of two low-energy conformations was

required for binding and that there is a requirement

for anticlockwise twist of the 5-phenyl ring relative to

the pyrimidine. 5-Phenyl-6-((3R,4S)-3,4,5-trihydroxy-

pentyl)pyrimidine-2,4-diamine 7a is shown here to be

an interesting lead compound for further evaluation

and further reﬁnement of design for optimisation of

potency and selectivity of inhibition of M. tuberculosis

DHFR and, hence, new approaches to treatment of this

widespread disease.

5. Conclusions

In this paper, we have reported our exploitation of a ma-

jor diﬀerence in the local structure in the region of the

dihydrofolate-binding sites of human and M. tuberculo-

sis DHFR to design a compound 7a which shows nota-

ble selectivity for inhibition of the latter. In the crystal

structure of a M. tuberculosis DHFR ternary complex

with methotrexate 2 and glycerol, the glycerol is held

tightly in its binding pocket by a network of ﬁve H-

bonds. This glycerol-binding pocket is close to the site

of the methotrexate. This glycerol-binding pocket is ab-

sent from the structure of human DHFR. In the struc-

tures of 7, the two-carbon link suggested by the crystal

structure joins a triol (mimicking the glycerol) to the

6-position of a pyrimidine-2,4-diamine core which binds

into the dihydrofolate-binding site. The conﬁgurations

of the secondary alcohols match the orientation of the

glycerol relative to the methotrexate in the crystal struc-

ture. Three series of analogues were also designed to test

the hypotheses of the design of 7. Compounds 8 tested

the assignment of the conﬁguration of the point of

attachment of the triol to the linker and hence to the

pyrimidine-2,4-diamine. Mono-hydroxy compounds 9

tested the need to take up the H bonds from all three

alcohols of the glycerol in binding selectively to the

6. Experimental

6.1. General

NMR spectra were recorded on JEOL/Varian GX270

and EX400 spectrometers of samples in CDCl₃, unless

otherwise stated. Mass spectra were obtained using a

VG7070E spectrometer. IR spectra were measured as

thin ﬁlms or as KBr discs on a Perkin-Elmer RXI FT-

IR spectrometer. Optical rotations were measured in a

10-cm cell on an Optical Activity Ltd. polarimeter; c is

expressed in grams per 100 mL. The stationary phase

for chromatography was silica gel. All reactions were

carried out under N₂at ambient temperature, unless

otherwise stated. Solvents were evaporated under re-

duced pressure. Melting points were determined by

using a Reichert-Jung Thermo Galen instrument and

are uncorrected.

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4563

6.2. 1-(4-Chlorophenyl)-1-cyano-2-methoxybut-1-ene (64)

and 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine (pyri-

methamine) (3)

(2H, d, J = 8.6 Hz, Ar 2,6-H₂), 7.44 (2H, d, J = 8.5 Hz,

Ar 3,5-H₂); NMR (CD₃OD) d_C30.05, 31.36, 62.70,

71.97, 74.14, 107.03, 129.18, 132.23, 133.13, 133.71,

161.73, 162.08, 163.06; MS m/z 341.1185 (M+H)

(C₁₅H₂₀³⁷ClN₄O₃requires 341.1194), 339.1225 (M+H)

Compound 62/63 was treated with CH₂N₂, as for the

synthesis of 24a, to give 64 (88%) as a pale yellow oil:

(C₁₅H₂₀³⁵ClN₄O₃

(MꢀCH₃OH), 249/247 (MꢀC₃H₇O₃).

requires

339.1223),

308/306

IR m_max2204, 1606 cm^ꢀ1

;

NMR 1.32 (3H, t,

J = 7.6 Hz, CMe), 2.80 (2H, q, J = 7.6 Hz, CH₂), 3.88

(3H, s, OMe), 7.31 (2H, d, J = 8.6 Hz, Ph 3,5-H₂), 7.61

(2H, d, J = 8.6 Hz, Ph 2,6-H₂). Compound 64 was trea-

ted with guanidine, as for the synthesis of 25a, to give 3

(50%) as a white solid: mp 233–235 ꢁC (lit.¹⁶mp 233–

234 ꢁC); NMR d_H0.97 (3H, t, J = 7.4 Hz, Me), 2.09

(2H, q, J = 7.4 Hz, CH₂), 5.64 (2H, br, NH₂), 5.92

(2H, br, NH₂), 7.22 (2H, d, J = 8.2 Hz, Ph 3,5-H₂),

7.49 (2H, d, J = 8.2 Hz, Ph 2,6-H₂); MS m/z 251.0884

(M+H) (C₁₂H₁₄³⁷ClN₄requires 251.0877), 249.0909

(M+H) (C₁₂H₁₄³⁵ClN₄requires 311.0910).

6.4.2. Method B. Compound 30b was treated with aq

CF₃CO₂H, as for the synthesis of 7a, to give 7b (91%)

as a white solid, with data as above.

6.5. 5-(4-Bromophenyl)-6-((3R,4S)-3,4,5-trihydroxypen-

tyl)pyrimidine-2,4-diamine (7c)

6.5.1. Method A. Compound 26c was treated with FeCl₃,

as for the synthesis of 7b, to give 7c (85%) as a white so-

20

lid: ½aꢁ ¼ ꢀ4:2^ꢂ(c 0.24, MeOH); mp 198–200 ꢁC;

D

NMR (D₂O) d_H1.47 (1H, m, 2⁰-H), 1.82 (1H, m, 2⁰-

H), 2.19 (1H, m, 1⁰-H), 2.33 (1H, m, 1⁰-H), 3.29 (1H,

m, 3⁰-H), 3.32–3.36 (2H, m, 5⁰-H₂), 3.48 (1H, m, 4⁰-

H), 7.08 (2H, d, J = 8.0 Hz, Ar 2,6-H₂), 7.55 (2H, d,

J = 8.0 Hz, Ar 3,5-H₂); MS m/z 385.0683 (M+H)

(C₁₅H₂₀⁸¹BrN₄O₃requires 385.0698), 383.0714 (M+H)

(C₁₅H₂₀⁷⁹BrN₄O₃requires 383.0718).

6.3. 5-Phenyl-6-((3R,4S)-3,4,5-trihydroxypentyl)pyrim-

idine-2,4-diamine (7a)

6.3.1. Method A. Compound 26a (150 mg, 0.4 mmol)

was treated with Na (84 mg, 3.6 mmol) in liquid NH₃

(10 mL) and THF (5 mL) at ꢀ33 ꢁC for 20 min. Satu-

rated aq NH₄Cl (2 mL) was added and the mixture

was allowed to warm to 20 ꢁC. CHCl₃(14 mL) and

MeOH (7 mL) were added and the mixture was ﬁltered.

Evaporation and chromatography (CHCl₃/MeOH, 7:3)

gave 7a (90 mg, 78%) as a white solid: mp 90–91 ꢁC;

NMR (D₂O) d_H1.29–1.36 (1H, m, 2-H), 1.49–1.55

(1H, m, 2-H), 2.07 (1H, ddd, J = 13.0, 10.2, 6.2 Hz, 1-

H), 2.21 (1H, ddd, J = 13.0, 10.5, 5.3 Hz, 1-H), 3.17–

3.21 (1H, m, 3-H), 3.22–3.25 (2H, m, 5-H₂), 3.37 (1H,

dt, J = 8.5, 6.1 Hz, 4-H), 7.03 (1H, d, J = 7.5 Hz, Ph 2-

H), 7.04 (1H, d, J = 7.5 Hz, Ph 6-H), 7.23 (1H, t,

J = 7.5 Hz, Ph 4-H), 7.29 (2H, t, J = 7.5 Hz, Ph 3,5-

H₂); NMR (D₂O) d_C30.24 (CH₂), 31.10 (CH₂), 62.25

(5-C), 71.35 (CH), 74.11 (CH), 109.19 (Pyr 5-C),

128.08 (Ph CH), 129.19 (2· Ph CH), 130.54 (2· Ph

CH), 133.88 (Ph 1-C), 161.15 (Pyr 2-C), 162.92 (Pyr 4-

C), 165.94 (Pyr 6-C); MS m/z 305.1616 (M+H)

(C₁₅H₂₁N₄O₃requires 305.1613), 327 (M+Na), 243

(MꢀC₂H₅O₂), 213 (MꢀC₃H₇O₃).

6.5.2. Method B. Compound 30c was treated with aq

CF₃CO₂H, as for the synthesis of 7a, to give 7c (87%)

as a pale yellow solid, with data as above.

6.6. 5-(3,4-Dichlorophenyl)-6-((3R,4S)-3,4,5-trihydroxy-

pentyl)pyrimidine-2,4-diamine (7d)

6.6.1. Method A. Compound 26d was treated with

FeCl₃, as for the synthesis of 7b, to give 7d (77%) as a

20

white solid: ½aꢁ ¼ ꢀ1:4^ꢂ(c 2.2, MeOH); mp 180–

D

181 ꢁC; NMR ((CD₃)₂SO) 1.43 (1H, m, 2⁰-H), 1.63 (1

H, m, 2⁰-H), 2.08 (1H, ddd, J = 13.6, 10.4, 5.5 Hz, 1⁰-

H), 2.34 (1H, ddd, J = 13.6, 10.4, 5.5 Hz, 1⁰-H), 3.17

(1H, m, 3⁰-H), 3.28–3.31 (2H, m, 5⁰-H₂), 3.48 (1H, m,

4⁰-H), 5.79 (2H, br, NH₂), 5.97 (2H, br, NH₂), 7.16

(1H, dd, J = 8.2, 1.8 Hz, Ar 6-H), 7.42 (1H, d,

J = 1.8 Hz, Ar 2-H), 7.66 (1H, d, J = 8.2 Hz, Ar 5-H);

NMR ((CD₃)₂SO) d_C31.26, 32.09, 63.94, 71.95, 75.19,

105.22, 131.38, 131.81, 133.16, 133.74, 133.97, 137.51,

6.3.2. Method B. Compound 30a was treated with aq

CF₃CO₂H, as for the synthesis of 25a (reaction time

6 h), to give 7a (85%) as a white solid, with data as

above.

162.42,

166.19;

MS

m/z

377.0794

(M+H)

(C₁₅H₁₉³⁷Cl₂N₄O₃requires 377.0775), 375.0814 (M+H)

37

(C₁₅H₁₉Cl³⁵ClN₄O₃requires 375.0804), 373.0836

(M+H) (C₁₅H₁₉³⁵Cl₂N₄O₃requires 373.0834), 345/343/

341 (MꢀCH₃O).

6.4. 5-(4-Chlorophenyl)-6-((3R,4S)-3,4,5-trihydroxy-

pentyl)pyrimidine-2,4-diamine (7b)

6.6.2. Method B. Compound 30d was treated with aq

CF₃CO₂H, as for the synthesis of 7a, to give 7d (77%)

as a white solid, with data as above.

6.4.1. Method A. Compound 26b (60 mg, 0.14 mmol)

was stirred with anhydrous FeCl₃(68 mg, 0.42 mmol)

in dry CH₂Cl₂(5 mL) under N₂for 80 min. Water

(2 mL) was added. Evaporation and chromatography

6.7. 5-Phenyl-6-((3S,4S)-3,4,5-trihydroxypentyl)pyrim-

idine-2,4-diamine (8a)

(CHCl₃/MeOH, 7:3) gave 7b (30 mg, 63%) as a white so-

20

D

lid: ½aꢁ ¼ ꢀ1:0^ꢂ(c 1.1, MeOH); mp 250–251 ꢁC; NMR

Compound 43a (200 mg, 0.5 mmol) was stirred in

MeOH (20 mL) with Pd/C (5%, 154 mg) and CHCl₃

(100 lL) under H₂for 2 h. Filtration (Celite^ꢂ), evapora-

tion and chromatography (CHCl₃/MeOH, 7:3) gave

8a (150 mg, 93%) as a white solid: mp > 350 ꢁC;

(D₂O) d_H1.44 (1H, m, 2⁰-H), 1.65 (1H, m, 2⁰-H), 2.22

(1H, ddd, J = 13.6, 10.4, 6.0 Hz, 1⁰-H), 2.36 (1H, ddd,

J = 13.6, 10.0, 5.2 Hz, 1⁰-H), 3.31 (1H, m, 3⁰-H), 3.35–

3.38 (2H, m, 5⁰-H₂), 3.48–3.53 (1H, m, 4⁰-H), 7.18

4564

20

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

½aꢁ ¼ þ10:9^ꢂ(c 0.5, H₂O); NMR (D₂O) d_H1.50-1.64

6.11. 1-(4-Chlorophenyl)-1-cyano-7-hydroxy-2-methoxy-

hept-1-ene (47b) and 5-(4-chlorophenyl)-6-(5-hydroxy-

pentyl)pyrimidine-2,4-diamine (9b)

D

(2H, m, 2⁰-H₂), 2.28 (1H, ddd, J = 13.9, 10.1, 6.3 Hz,

1⁰-H), 2.40 (1H, ddd, J = 13.9, 10.1, 6.3 Hz, 1⁰-H),

3.31–3.45 (4H, m, 3⁰,4⁰,5⁰-H₄), 7.25 (2H, d, J = 7.0 Hz,

Ph 2,6-H₂), 7.42 (1H, t, J = 7.0 Hz, Ph 4-H), 7.47 (2H,

t, J = 7.0 Hz, Ph 3,5-H₂); NMR (D₂O) d_C29.75, 31.57,

62.63, 73.61, 73.64, 109.24, 128.37, 129.37, 130.56,

133.41, 160.29, 161.05, 161.81; MS m/z 305.1618

(M+H) (C₁₅H₂₁N₄O₃requires 305.1613).

Compound 45b/46b was treated with CH₂N₂, as for the

synthesis of 24a (followed by chromatography (EtOAc/

hexane, 3:1)), to give 47b (62%) as a pale yellow oil:

NMR d_H1.52–1.80 (6 H, m, 4,5,6-H₆), 2.77 (2H, t,

J = 7.8 Hz, 3-H₂), 3.68 (2H, t, J = 6.2 Hz, 7-H₂), 3.85

(3H, s, Me), 7.29 (2H, d, J = 8.6 Hz, Ar 2,6-H₂), 7.54

(2H, d, J = 8.6 Hz, Ar 3,5-H₂); MS m/z 282.1077

(M+H) (C₁₅H₁₉³⁷ClNO₂requires 280.1074), 280.1102

(M+H) (C₁₅H₁₉³⁵ClNO₂requires 280.1104), 264/262

(MꢀOH). Compound 47b was treated with guanidine,

as for the synthesis of 9a, to give 9b (59%) as a white

solid: mp 165–166 ꢁC; IR m_max3407, 3329, 3174,

6.8. 5-(4-Chlorophenyl)-6-((3S,4S)-3,4,5-trihydroxypen-

tyl)pyrimidine-2,4-diamine (8b)

Compound 43b was treated with FeCl₃, as for the syn-

thesis of 7b, to give 8b (77%) as a white solid:

20

D

mp > 350 ꢁC; ½aꢁ ¼ þ6:0^ꢂ(c 0.67, H₂O); NMR

(CD₃OD) d_H1.73–1.77 (2H, m, 2⁰-H₂), 2.41 (1H, dt,

J = 14.4, 6.7 Hz, 1⁰-H), 2.54 (1H, dt, J = 14.4, 6.7 Hz,

1⁰-H), 3.44–3.61 (4H, m, 3⁰,4⁰,5⁰-H₄), 7.31 (2H, d,

J = 8.4 Hz, Ar 2,6-H₂), 7.53 (2H, d, J = 8.4 Hz, Ar 3,5-

H₂); NMR (CD₃OD) d_C27.08, 31.39, 62.91, 70.52,

73.58, 108.29, 129.60, 132.04, 137.45, 137.50, 156.86,

1631 cm^ꢀ1

; NMR ((CD₃)₂SO) d_H1.11 (2H, qn,

J = 7.4 Hz, 3⁰-H₂), 1.26 (2H, qn, J = 7.4 Hz, 4⁰-H₂),

1.42 (2H, qn, J = 7.4 Hz, 2⁰-H₂), 2.07 (2H, t,

J = 7.4 Hz, 1⁰-H₂), 3.29 (2H, t, J = 7.4 Hz, 5⁰-H₂), 4.29

(1H, br, OH), 5.64 (2H, br, NH₂), 5.94 (2H, br, NH₂)

7.18 (2H, d, J = 8.2 Hz, Ar 2,6-H₂), 7.47 (2H, d,

J = 8.2 Hz, Ar 3,5-H₂); NMR ((CD₃)₂SO) d_C25.88,

28.58, 32.69, 34.63, 61.00, 106.16, 129.34, 132.25,

134.76, 133.10, 135.47, 162.44, 162.47, 165.87; MS m/z

309.1310 (M+H) (C₁₅H₂₀³⁷ClN₄O₂requires 309.1296),

307.1335 (M+H) (C₁₅H₂₀³⁵ClN₄O₂requires 307.1325),

236/234 (MꢀC₄H₈O).

157.38,

157.80;

MS

m/z

341.1180

(M+H)

(C₁₅H₂₀³⁷ClN₄O₃requires 341.1194), 339.1237 (M+H)

(C₁₅H₂₀³⁵ClN₄O₃requires 339.1223).

6.9. 5-(4-Bromophenyl)-6-((3S,4S)-3,4,5-trihydroxypen-

tyl)pyrimidine-2,4-diamine (8c)

Compound 43c was treated with FeCl₃, as for the

synthesis of 7b, to give 8c (77%) as a white solid:

6.12. 1-(4-Bromophenyl)-1-cyano-7-hydroxy-2-methoxy-

hept-1-ene (47c) and 5-(4-bromophenyl)-6-(5-hydroxy-

pentyl)pyrimidine-2,4-diamine (9c)

20

mp > 350 ꢁC; ½aꢁ ¼ þ12:5^ꢂ(c 0.24, MeOH); IR m_max

D

3649, 3468, 3418, 1618 cm^ꢀ1; NMR (D₂O) d_H1.60–

1.72 (2H, m, 2⁰-H₂), 2.30–2.54 (2H, m, 1⁰-H₂), 3.42–

3.56 (4H, m, 3⁰,4⁰,5⁰-H₄), 7.25 (2H, d, J = 8.7 Hz,

Ar 2,6-H₂), 7.72 (2H, d, J = 8.7 Hz, Ar 3,5-H₂); MS

m/z 385.0707 (M+H) (C₁₅H₂₀⁸¹BrN₄O₃requires

385.0698), 383.0717 (M+H) (C₁₅H₂₀⁷⁹BrN₄O₃requires

383.0718).

Compound 45c/46c was treated with CH₂N₂, as for the

synthesis of 47b, to give 47c (32%) as a pale yellow oil:

NMR d_H1.53-1.75 (6 H, m, 4,5,6-H₆), 2.76 (2H, t,

J = 7.0 Hz, 3-H₂), 3.69 (2H, t, J = 7.0 Hz, 7-H₂), 3.85

(3H, s, Me), 7.44 (2H, d, J = 8.8 Hz, Ar 2,6-H₂), 7.48

(2H, d, J = 8.8 Hz, Ar 3,5-H₂); MS m/z 326.0583

(M+H) (C₁₅H₁₉⁸¹BrNO₂requires 326.0578), 324.0596

(M+H) (C₁₅H₁₉⁷⁹BrNO₂requires 324.0599). Compound

47c was treated with guanidine, as for the synthesis of

9a, to give 9c (43%) as a white solid: mp 177–178 ꢁC;

IR m_max3550, 3468, 3414, 1617 cm^ꢀ1; NMR ((CD₃)₂SO)

d_H1.11 (2H, qn, J = 7.4 Hz, 3⁰-H₂), 1.25 (2H, qn,

J = 7.4 Hz, 4⁰-H₂), 1.42 (2H, qn, J = 7.4 Hz, 2⁰-H₂),

2.07 (2H, t, J = 7.4 Hz, 1⁰-H₂), 3.27 (2H, t, J = 6.4 Hz,

5⁰-H₂), 4.30 (1H, br, OH), 5.74 (2H, br, NH₂), 6.00

(2H, br, NH₂), 7.11 (2H, d, J = 8.4 Hz, Ar 2,6-H₂),

7.57 (2H, d, J = 8.4 Hz, Ar 3,5-H₂); NMR (CF₃CO₂H

salt) ((CD₃)₂SO) d_C25.33, 27.72, 30.33, 32.21, 60.68,

108.00, 115.78 (q, J = 289.1 Hz), 122.75, 130.74,

132.74, 133.24, 153.43, 155.31, 158 (q, J = 37.6 Hz),

164.30; MS m/z 353.0807 (M+H) (C₁₅H₂₀⁸¹BrN₄O

requires 353.0800), 351.0816 (M+H) (C₁₅H₂₀⁷⁹BrN₄O

requires 351.0820).

6.10. 1-Cyano-7-hydroxy-2-methoxy-1-phenylhept-1-ene

(47a) and 6-(5-hydroxypentyl)-5-phenylpyrimidine-2,4-

diamine (9a)

Compound 45a/46a was treated with CH₂N₂, as for the

synthesis of 24a (followed by chromatography (EtOAc/

hexane, 2:1)), to give 47a (78%) as a pale yellow oil: IR

m_max3439, 2204 cm^ꢀ1

; MS m/z 246.1492 (M+H)

(C₁₅H₂₀NO₂requires 246.1494). Compound 47a was

treated with guanidine, as for the synthesis of 25a (chro-

matographic eluant CH₂Cl₂/MeOH, 4:1), to give 9a

(36%) as a white solid: mp 214–216 ꢁC; IR m_max3420,

3331, 3177, 1619 cm^ꢀ1; NMR d_H1.27 (2H, qn,

J = 7.2 Hz, 3⁰-H₂), 1.45 (2H, qn, J = 7.2 Hz, 4⁰-H₂),

1.55 (2H, qn, J = 7.2 Hz, 2⁰-H₂), 2.28 (2H, t,

J = 7.2 Hz, 1⁰-H₂), 3.56 (2H, t, J = 6.4 Hz, 5⁰-H₂), 4.59

(2H, br, NH₂), 4.98 (2 H, br, NH₂), 7.21 (2H, d,

J = 7.2 Hz, Ph 2,6-H₂), 7.37 (1H, t, J = 7.2 Hz, Ph 4-

H), 7.44 (2H, t, J = 7.2 Hz, Ph 3,5-H₂); NMR (CD₃OD)

d_C25.38, 28.49, 31.81, 33.79, 61.33, 108.29, 127.67,

128.95, 130.51, 134.76, 161.32, 162.98, 165.09; MS m/z

273.1704 (M+H) (C₁₅H₂₀N₄O requires 273.1715), 213

(MꢀC₃H₇O), 200 (MꢀC₄H₈O).

6.13. 1-Cyano-1-(3,4-dichlorophenyl)-7-hydroxy-2-meth-

oxyhept-1-ene (47d) and 5-(3,4-dichlorophenyl)-6-(5-

hydroxypentyl)pyrimidine-2,4-diamine (9d)

Compound 45c/46c was treated with CH₂N₂, as for the

synthesis of 47b, to give 47d (68%) as a pale yellow oil:

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4565

NMR d_H1.41–1.50 (4H, m, 5,6-H₄), 1.62 (2H, qn,

J = 7.6 Hz, 4-H₂), 2.75 (2H, t, J = 7.6 Hz, 3-H₂), 3.41

(2H, t, J = 6.0 Hz, 7-H₂), 3.95 (3H, s, Me), 7.50 (1H, dd,

J = 8.6, 2.0 Hz, Ar 6-H), 7.63 (1H, d, J = 8.6 Hz, Ar 5-

H), 7.75 (1H, d, J = 2.0 Hz, Ar 2-H); MS m/z 318.0686

(M+H) (C₁₅H₁₈³⁷Cl₂NO₂requires 318.0655), 316.0689

(M+H) (C₁₅H₁₈³⁷Cl³⁵ClNO₂requires 316.0685),

314.0715 (M+H) (C₁₅H₁₈³⁵Cl₂NO₂requires 314.0714),

291/289/287 (MꢀCN). Compound 47d was treated with

guanidine, as for the synthesis of 9a, to give 9d (43%) as

a white solid: mp 94–95 ꢁC; IR m_max3499, 3419, 3333,

(95%) as a highly hygroscopic pale yellow solid:

20

½aꢁ ¼ ꢀ15^ꢂ(c 0.9, MeOH); IR m_max3550, 3478, 3414,

D

1618 cm^ꢀ1; NMR (CD₃CN) d_H3.51 (1H, dd, J = 12.3,

4.9 Hz, 3⁰-H), 3.55 (1H, dd, J = 12.3, 4.5 Hz, 3⁰-H),

3.74 (1H, m, 2⁰-H), 4.49 (1H, d, J = 6.2 Hz, 1⁰-H), 5.94

(2H, br, NH₂), 7.03 (2H, br, NH₂), 7.29 (2H, d,

J = 8.0 Hz, Ar 2,6-H₂), 7.71 (2H, d, J = 8.0 Hz, Ar 3,5-

H₂); NMR (CD₃CN) d_C62.47, 68.98, 72.30, 108.83,

123.37, 129.70, 132.92, 132.98, 133.81, 156.05, 162.06,

164.91; MS m/z 379/377 (M+Na), 357.0396 (M+H)

(C₁₃H₁₆⁸¹BrN₄O₃requires 357.0385), 355.0412 (M+H)

(C₁₃H₁₆⁷⁹BrN₄O₃requires 355.0405).

1622 cm^ꢀ1

; NMR ((CD₃)₂SO) d_H1.12 (2H, qn,

J = 7.4 Hz, 3⁰-H₂), 1.25 (2H, qn, J = 7.4 Hz, 4⁰-H₂), 1.42

(2H, qn, J = 7.4 Hz, 2⁰-H₂), 2.07 (2H, t, J = 7.4 Hz, 1⁰-

H₂), 3.27 (2H, q, J = 5.6 Hz, 5⁰-H₂), 4.28 (1H, t,

J = 5.6 Hz, OH), 5.72 (2H, br, NH₂), 5.90 (2H, br,

NH₂), 7.11 (1H, dd, J = 8.2, 2.2 Hz, Ar 6-H), 7.36 (1H,

d, J = 2.2 Hz, Ar 2-H), 7.62 (1 H, d, J = 8.2 Hz, Ar 5-

H); NMR ((CD₃)₂SO) d_C25.87, 28.50, 32.70, 34.64,

61.02, 105.23, 130.21, 131.37, 131.74, 131.81, 133.19,

137.66, 162.36, 162.69, 165.96; MS m/z 345.0885 (M+H)

(C₁₅H₁₉³⁷Cl₂N₄O requires 345.0876), 343.0901 (M+H)

(C₁₅H₁₉³⁷Cl³⁵ClN₄O requires 343.0906), 341.0927

(M+H) (C₁₅H₁₉³⁵Cl₂N₄O requires 341.0935), 285/283/

281 (MꢀC₃H₇O), 272/270/268 (MꢀC₄H₈O).

6.17. 5-(3,4-Dichlorophenyl)-6-((1S,2R)-1,2,3-trihydroxy-

propyl)pyrimidine-2,4-diamine (10d)

Compound 52d was treated with aq CF₃CO₂H, as for

the synthesis of 10c, to give 10d (87%) as a pale yellow

20

solid: mp 120–121 ꢁC; ½aꢁ ¼ ꢀ3:0^ꢂ(c 4.7, MeOH); IR

D

m_max3549, 3476, 3415, 1618 cm^ꢀ1; NMR (CD₃CN) d_H

3.41 (1H, d, J = 13.1 Hz, 3⁰-H), 3.45 (1H, d,

J = 13.1 Hz, 3⁰-H), 3.63–3.68 (1H, m, 2⁰-H), 4.35 (1H,

d, J = 4.7 Hz, 1⁰-H), 5.25 (2H, br, NH₂), 5.67 (2H, br,

NH₂), 7.20 (1H, dd, J = 8.0, 1.9 Hz, Ar 6-H), 7.46

(1H, d, J = 1.9 Hz, Ar 2-H), 7.60 (1H, d, J = 8.0 Hz,

Ar 5-H); NMR ((CD₃)₂SO) d_C63.67, 69.38, 74.29,

106.16, 130.30, 131.08, 131.44, 131.63, 132.68, 134.09,

162.01, 162.81, 164.00; MS m/z 371/369/367 (M+Na),

349.0469 (M+H) (C₁₃H₁₅³⁷Cl₂N₄O₃requires 349.0462),

347.0501 (M+H) (C₁₃H₁₅³⁷Cl³⁵ClN₄O₃requires

347.0491), 345.0521 (M+H) (C₁₃H₁₅³⁵Cl₂N₄O₃requires

345.0521).

6.14. 5-Phenyl-6-((1S,2R)-1,2,3-trihydroxypropyl)pyrim-

idine-2,4-diamine (10a)

Compound 52a was treated with aq CF₃CO₂H, as for the

synthesis of 26a (reaction time 2 h), to give 10a (73%) as a

20

D

highly hygroscopic white solid: ½aꢁ ¼ ꢀ0:38^ꢂ(c 4,

MeOH); NMR (CD₃CN) d_H3.45 (1H, dd, J = 11.6,

4.9 Hz, 3⁰-H), 3.48 (1H, dd, J = 11.6, 3.9 Hz, 3⁰-H),

3.72 (1H, m, 2⁰-H), 4.46 (1H, d, J = 6.2 Hz, 1⁰-H), 4.73

(2H, br, NH₂), 5.82 (1H, br, NH), 6.98 (1H, br, NH),

7.31 (2H, dd, J = 7.4, 2.0 Hz, Ph 2,6-H₂), 7.45-7.61

(3H, m, Ph 3,4,5-H₃); MS m/z 299 (M + Na), 277.1308

(M+H) (C₁₃H₁₇N₄O₃requires 277.1300).

6.18. 1-Cyano-1,4-diphenyl-2-methoxybut-1-ene (56) and

5-phenyl-6-(2-phenylethyl)pyrimidine-2,4-diamine (11)

Compound 54/55 was treated with CH₂N₂, as for the

synthesis of 24a, to give 56 (95%) as a pale yellow oil:

IR m_max2204 cm^ꢀ1

;

MS m/z 264.1390 (M+H)

(C₁₈H₁₈NO requires 264.1388), 236 (MꢀHCN), 91

(Bn). Compound 56 was treated with guanidine, as for

the synthesis of 25a (chromatographic eluant CH₂Cl₂/

MeOH, 8:1), to give 11 (32%) as a pale yellow solid:

mp 116–118 ꢁC; NMR d_H2.54 (2H, t, J = 8.0 Hz,

CH₂), 2.83 (2H, t, J = 8.0 Hz, CH₂), 4.62 (2H, br,

NH₂), 4.99 (2H, br, NH₂), 6.94 (2H, d, J = 6.8 Hz, Ph

2,6-H₂), 7.05 (2H, d, J = 6.5 Hz, Ph⁰2,6-H₂), 7.14 (1H,

t, J = 6.8 Hz, Ph 4-H), 7.17 (2H, t, J = 6.8 Hz, Ph 3,5-

H₂) 7.35 (2H, t, J = 6.5 Hz, Ph⁰4-H), 7.39 (2H, t,

J = 6.5 Hz, Ph⁰3,5-H₂); MS m/z 291.1616 (M+H)

(C₁₈H₁₉N₄requires 291.1609), 199 (MꢀBn).

6.15. 5-(4-Chlorophenyl)-6-((1S,2R)-1,2,3-trihydroxy-

propyl)pyrimidine-2,4-diamine (10b)

Compound 52b was treated with aq CF₃CO₂H, as for

the synthesis of 26a, to give 10b (90%) as a pale yellow

20

solid: mp 196–197 ꢁC; ½aꢁ ¼ ꢀ41^ꢂ(c 0.4, MeOH); IR

D

m_max3550, 3475, 3413, 1617 cm^ꢀ1; NMR ((CD₃)₂SO)

d_H3.58 (1H, dd, J = 11.3, 4.7 Hz, 3⁰-H, 3.63 (1H, dd,

J = 11.3, 3.5 Hz, 3⁰-H), 3.89–3.92 (1H, m, 2⁰-H), 4.54

(1H, d, J = 7.0 Hz, 1⁰-H), 5.54 (5H, br) and, 6.62 (1H,

br) (2· NH₂+ 2· OH), 7.40 (2H, d, J = 7.4 Hz, Ar

2,6-H₂), 7.47 (2H, d, J = 7.4 Hz, Ar 3,5-H₂),7.79 (1H,

br, OH); NMR ((CD₃)₂SO) d_C62.60, 69.31, 72.47,

108.42, 129.88, 132.39, 133.53, 134.72, 161.21, 161.53,

161.87; MS m/z 313.0877 (M+H) (C₁₃H₁₆³⁷ClN₄O₃re-

quires 313.0881), 311.0905 (M+H) (C₁₃H₁₆³⁵ClN₄O₃re-

quires 311.0910).

6.19. 1-Cyano-2-methoxy-1-phenylprop-1-ene (60) and

6-methyl-5-phenylpyrimidine-2,4-diamine (12)

Compound 58/59 was treated with CH₂N₂, as for the

synthesis of 24a, to give 60 (87%) as a pale yellow oil:

IR m_max2204, 1606 cm^ꢀ1; NMR d_H2.45 (3H, s, CMe),

3.85 (3H, s, OMe), 7.26 (1H, t, J = 7.0 Hz, Ph 4-H),

7.30 (2H, t, J = 7.0 Hz, Ph 3,5-H₂), 7.61 (2H, t,

J = 7.0 Hz, Ph 2,6-H₂); MS m/z 174.0921 (M+H)

(C₁₁H₁₂NO requires 174.0918). Compound 60 was

treated with guanidine, as for the synthesis of 25a

6.16. 5-(4-Bromophenyl)-6-((1S,2R)-1,2,3-trihydroxy-

propyl)pyrimidine-2,4-diamine (10c)

Compound 52c was treated with aq CF₃CO₂H, as for

the synthesis of 26a (reaction time 4 h), to give 10c

4566

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

(chromatographic eluant CH₂Cl₂/MeOH, 4:1), to give

12 (38%) as a pale yellow solid: mp 250–251 ꢁC (lit.¹⁹

mp 249–251 ꢁC); IR m_max3395, 3323 cm^ꢀ1; NMR

((CD₃)₂SO) d_H1.85 (3H, s, Me), 5.62 (2H, br, NH₂),

6.00 (2H, br, NH₂), 7.20 (2H, d, J = 7.3 Hz, Ph 2,6-

H₂), 7.33 (1H, t, J = 7.3 Hz, Ph 4-H), 7.43 (2H, t,

J = 7.3 Hz, Ph 3,5-H₂); MS m/z 201.1145 (M+H)

(C₁₁H₁₃N₄requires 201.1140), 123 (MꢀC₆H₅), 109

(MꢀC₇H₇).

3 h. Filtration (Celite^ꢂ) and evaporation gave 20 (2.3 g,

99%) as a pale yellow oil (lit.⁴⁶oil): NMR d_H1.26 (3H, t,

J = 7.0 Hz, CH₂CH₃), 1.33 (3H, s, 2-Me), 1.42 (3H, s, 2-

Me), 1.82 (2H, m, CH₂CH₂CO₂), 2.39 (1H, br, OH),

2.40 (1H, dt, J = 16.4, 7.8 Hz, CHCO₂), 2.53 (1H, dt,

J = 16.4, 7.4 Hz, CHCO₂), 3.65 (2H, d, J = 5.1 Hz,

CH₂OH), 4.09–4.20 (4 H, m, 4-H + 5-H + CH₂CH₃);

MS m/z 233.1396 (M+H) (C₁₁H₁₉O₅requires

233.1388), 217 (MꢀCH₃).

6.20. 2,3-O-Isopropylidene-^L-erythrose (18)

6.23. Ethyl (4S,5R)-4-benzyloxymethyl-2,2-dimethyl-1,3-

dioxolane-4-propanoate (21)

L-Arabinose 17 (10.0 g, 67 mmol), TsOHÆH₂O (150 mg,

0.79 mmol)

and

2,2-dimethoxypropane

(23.0 g,

LiN(SiMe₃)₂(1.0 M in THF, 10 mL, 10 mmol) was stir-

red with 20 (2.3 g, 10 mmol) and BnBr (3.4 g, 20 mmol)

in dry DMF (5 mL). After 2 h, water was added. The

mixture was extracted (Et₂O). The extract was washed

with water and brine and was dried. Evaporation and

221 mol) were stirred in dry DMF (130 mL) under N₂

for 90 min. The mixture was neutralised with Na₂CO₃.

The evaporation residue was added to water (120 mL)

and hexane (60 mL). NaIO₄(35.5 g, 0.17 mol) was

added to the aq layer and the mixture was stirred for

2 h. Na₂CO₃was added and the slurry was stirred for

1 h. The mixture was extracted with EtOAc. Evapora-

tion and chromatography (Et₂O/hexane, 2:1) gave 18

(5.8 g, 54%) as a colourless oil (lit.⁴⁵oil): NMR d_H

1.31 (3 H, s, Me), 1.46 (3H, s, Me), 3.89 (1H, d,

J = 2.5 Hz, OH), 4.01 (1H, d, J = 10.5 Hz, 4-H), 4.05

(1H, dd, J = 10.5, 3.5 Hz, 4-H), 4.55 (1H, d,

J = 6.0 Hz, 2-H), 4.82 (1H, dd, J = 6.0, 3.5 Hz, 3-H),

5.39 (1H, d, J = 2.5 Hz, 1-H); MS m/z 181 (M+Na),

159.0650 (MꢀH) (C₇H₁₁O₄requires 159.0657).

chromatography (Et₂O/hexane, 1:4) aﬀorded 21 (1.6 g,

20

D

48%) as a pale yellow oil: ½aꢁ ¼ þ24:8^ꢂ(c 4.4, CHCl₃);

NMR d_H1.24 (3H, t, J = 7.0 Hz, CH₂CH₃), 1.33 (3H, s,

2-Me), 1.42 (3H, s, 2-Me), 1.72–1.86 (2H, m,

CH₂CH₂CO₂), 2.50 (1H, m, CHCO₂), 2.48–2.54 (1H,

m, CHCO₂), 3.50 (2H, m, CH₂OBn), 4.08–4.15 (3H,

m, 5-H+CH₂CH₃), 4.28 (1H, dd, J = 11.9, 6.1 Hz, 4-

H), 4.50 (1H, d, J = 12.1 Hz, CHPh), 4.57 (1H, d,

J = 12.1 Hz, CHPh), 7.24–7.33 (5H, m, Ph-H₅); MS m/

z 323.1864 (M+H) (C₁₈H₂₇O₅requires 323.1858), 265

(MꢀC₃H₅O), 91 (Bn).

6.21. Ethyl (Z,4S,5R)-4-hydroxymethyl-2,2-dimethyl-1,3-

dioxolane-5-propenoate (19Z) and ethyl (E,4S,5R)-4-

hydroxymethyl-2,2-dimethyl-1,3-dioxolane-5-propenoate

(19E)

6.24. (4R,5S)-5-Benzyloxymethyl-4-(4-cyano-3-oxo-4-

phenylbutyl)-2,2-dimethyl-1,3-dioxolane (22a)/(4R,5S)-5-

benzyloxymethyl-4-(4-cyano-3-hydroxy-4-phenylbut-3-

enyl)-2,2-dimethyl-1,3-dioxolane (23a)

Ethyl triphenylphosphoranylidineacetate (9.3 g, 27 mmol)

was stirred with 18 (2.9 g, 18 mmol) in CH₂Cl₂

(130 mL) for 16 h. The evaporation residue was ex-

tracted with Et₂O. Evaporation and chromatography

(Et₂O/hexane, 1:1) gave 19Z (2.2 g, 54%) as a colourless

oil (lit.⁴⁶oil): NMR d_H1.29 (3 H, t, J = 7.0 Hz,

CH₂CH₃), 1.40 (3H, s, 2-Me), 1.53 (3H, s, 2-Me), 2.44

(1H, dd, J = 7.4, 5.5 Hz, OH), 3.45 (1H, m, CHHOH),

3.59 (1H, m, CHHOH), 4.16 (2H, q, J = 7.4 Hz,

CH₂CH₃), 4.53–4.57 (1H, m, 4-H), 5.58 (1H, dt,

J = 7.1, 1.7 Hz, 5-H), 5.91 (1H, dd, J = 11.7, 1.7 Hz,

CHCO₂), 6.36 (1H, dd, J = 11.7, 7.1 Hz, CH@CCO₂).

Further elution gave 19E (600 mg, 15%) as a colourless

oil (lit.⁴⁶oil): NMR d_H1.29 (3H, t, J = 7.2 Hz,

CH₂CH₃), 1.40 (3H, s, 2-Me), 1.52 (3H, s, 2-Me), 2.41

(1 H, t, J = 5.9 Hz, OH), 3.55 (2H, t, J = 5.9 Hz,

CH₂OH), 4.18 (2H, q, J = 7.2 Hz, CH₂CH₃), 4.35 (1H,

m, 4-H), 4.79 (1H, dt, J = 5.5, 1.6 Hz, 5-H), 6.12 (1H,

dd, J = 15.6, 1.6 Hz, CHCO₂), 6.88 (1H, dd, J = 15.6,

5.5 Hz, CHC@CCO₂); MS m/z 231.1240 (M+H)

(C₁₁H₁₉O₅requires 231.1232), 215 (MꢀCH₃), 173

(MꢀC₃H₅O), 143 (M – C₄H₇O₂).

LiN(SiMe₃)₂(1.0 M in THF, 9.1 mL, 9.1 mmol) was

added to phenylacetonitrile (1.1 g, 9.4 mmol) in dry

Et₂O (10 mL) under N₂at ꢀ78 ꢁC. After 10 min, 21

(2.9 g, 9.0 mmol) was added. The mixture was allowed

to warm to 20 ꢁC and was stirred for 72 h. Water was

added. The solution was washed twice (Et₂O) before

being acidiﬁed to pH 6 with aq citric acid (1 M) in the

presence of EtOAc. The EtOAc phase was separated

and washed with water. Drying, evaporation and chro-

matography (EtOAc/hexane, 2:1) gave 22a/23a (1.5 g,

21%) as a yellow oil: IR m_max2207, 1728 cm^ꢀ1; NMR

d_H1.26 (3H, s, 2-Me), 1.34 (3H, s, 2-Me), 1.66–1.80

(2H, m, CH₂CH₂CO), 2.63 (1H, m, CHCO), 2.75–2.80

(1H, m, CHCO), 3.45 (2H, d, J = 6.0 Hz, CH₂OBn),

3.98 (1H, m, 4-H), 4.20 (1H, q, J = 6.0 Hz, 5-H), 4.45

(1H, d, J = 11.5 Hz, CHPh), 4.52 (1H, d, J = 11.5 Hz,

CHPh), 7.21–7.41 (10 H, m, 2· Ph-H₅), 8.98 (1H, s,

OH); MS m/z 394.2016 (M+H) (C₂₄H₂₈NO₄requires

394.2018), 91 (Bn).

6.25. (4R,5S)-5-Benzyloxymethyl-4-(4-(4-chlorophenyl)-

4-cyano-3-oxobutyl)-2,2-dimethyl-1,3-dioxolane (22b)/

(4R,5S)-5-benzyloxymethyl-4-(4-cyano-3-hydroxy-4-(4-

chlorophenyl)but-3-enyl)-2,2-dimethyl-1,3-dioxolane (23b)

6.22. Ethyl (4S,5R)-4-hydroxymethyl-2,3-dimethyl-1,3-

dioxolane-5-propanoate (20)

4-Chlorophenylacetonitrile and 21 were condensed as

for the synthesis of 22a/23a (chromatographic eluant

EtOAc/hexane, 1:1), to give 22b/23b (26%) as a yellow

A mixture of 19Z and 19E (2.3 g, 10 mmol) was stirred

in EtOH (100 mL) with Pd/C (5%, 150 mg) under H₂for

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4567

oil: NMR d_H1.25 (2.7H, s, Me), 1.34 (2.7H, s, Me),

1.40 (0.3 H, s, Me), 1.51 (0.3H, s, Me), 1.67-1.88 (2H,

m, CH₂CH₂CO), 1.98–2.08 (0.2H, m, CH₂CO), 2.60–

2.80 (1.8 H, m, CH₂CO), 3.45 (1H, dd, J = 12.1,

6.0 Hz, CHOBn), 3.47 (1H, dd, J = 12.1, 6.0 Hz,

CHOBn), 4.00 (0.9H, ddd, J = 8.6, 6.0, 2.3 Hz, 4-H),

4.22 (0.9H, q, J = 6.0 Hz, 5-H), 4.29 (0.1H, ddd,

J = 10.14, 6.0, 3.9 Hz, 4-H), 4.39 (0.1H, q, J = 6.0 Hz,

5-H), 4.45 (0.1H, d, J = 11.5 Hz, CHPh), 4.49 (0.1H,

d, J = 11.5 Hz, CHPh), 4.51 (0.9H, d, J = 11.9 Hz,

CHPh), 4.56 (0.9H, d, J = 11.9 Hz, CHPh), 5.52

(0.1H, s, CHCN), 7.16 (0.2H, d, J = 8.6 Hz, Ar 2,6-

H₂), 7.25–7.32 (5H, m, Ph-H₅), 7.35 (1.8H, d,

J = 8.6 Hz, Ar 3,5-H₂), 7.84 (1.8H, d, J = 8.6 Hz, Ar

2,6-H₂), 9.33 (0.9H, br, OH); MS m/z 430.1604

(M+H) (C₂₄H₂₆³⁷ClNO₄requires 430.1599), 428.1618

(M+H) (C₂₄H₂₇³⁵ClNO₄requires 428.1628), 370

(MꢀC₂H₄NO), 91 (Bn).

6.28. (4R,5S)-5-Benzyloxymethyl-4-(4-cyano-3-methoxy-

4-phenylbut-3-enyl)-2,2-dimethyl-1,3-dioxolane (24a) and

6-(2-((4R,5S)-5-benzyloxymethyl-2,2-dimethyl-1,3-dioxo-

lan-4-yl)ethyl)-5-phenylpyrimidine-2,4-diamine (25a)

Compound 22a/23a (1.5 g, 3.7 mmol) in THF (5 mL)

was treated with CH₂N₂(8.0 mmol) in Et₂O (20 mL)

at 10 ꢁC for 16 h. Excess CH₂N₂was destroyed by care-

ful addition of AcOH (30% in THF). Evaporation gave

24a (1.2 g, 81%) as a yellow oil: NMR d_H1.37 (3H, s, 2-

Me), 1.46 (3H, s, 2-Me), 1.77–1.88 (2H, m, CH₂CHO),

2.78–2.86 (1H, m, CHC@C), 2.90–2.98 (1H, m,

CHC@C), 3.53 (2H, d, J = 5.9 Hz, CH₂OBn), 3.78

(3H, s, OMe), 4.22 (1H, m, 4-H), 4.33 (1H, m, 5-H),

4.51 (1H, d, J = 12.1 Hz, CHPh), 4.59 (1H, d,

J = 12.1 Hz, CHPh), 7.21–7.59 (10 H, m, 2· Ph-H₅);

MS m/z 408.2166 (M+H) (C₂₅H₃₀NO₄requires

408.2174), 350 (MꢀC₂H₃NO), 91 (Bn). NaOMe (140

g, 2.6 mmol) was stirred with guanidine.HCl (300 mg,

2.6 mmol) in MeO(CH₂)₂OH (10 mL) for 5 min at

30 ꢁC. The ﬁltered solution was boiled under reﬂux with

24a (700 mg, 1.8 mmol) for 16 h. Evaporation and chro-

matography (CHCl₃/MeOH, 19:1) gave 25a (400 mg,

46%) as a highly hygroscopic pale yellow solid: IR m_max

3415, 1685 cm^ꢀ1; NMR d_H1.23 (3H, s, Me), 1.24 (3H, s,

Me), 1.61–1.67 (2H, m, CH₂CHO), 2.24 (1H, m, Pyr-

CH), 2.51 (1H, m, Pyr-CH), 3.4 (2H, d, J = 6.0 Hz,

CH₂OBn), 3.95-4.00 (1H, m, dioxolane 4-H), 4.15 (1H,

q, J = 6.0 Hz, dioxolane 5-H), 4.44 (1H, d,

J = 12.3 Hz, CHPh), 4.53 (1H, d, J = 12.3 Hz, CHPh),

4.68 (2H, br, NH₂), 5.01 (2H, br, NH₂), 7.09–7.39

(10H, m, 2· Ph-H₅); MS m/z 435.2423 (M+H)

(C₂₅H₃₁N₄O₃requires 435.2396), 200 (MꢀC₁₄H₁₈O₃),

91 (Bn).

6.26. (4R,5S)-5-Benzyloxymethyl-4-(4-(4-bromophenyl)-

4-cyano-3-oxobutyl)-2,2-dimethyl-1,3-dioxolane (22c)/

(4R,5S)-5-benzyloxymethyl-4-(4-cyano-3-hydroxy-4-(4-

bromophenyl)but-3-enyl)-2,2-dimethyl-1,3-dioxolane

(23c)

4-Bromophenylacetonitrile and 21 were condensed, as

for the synthesis of 22a/23a, to give 22c/23c (18%) as a

pale yellow oil: NMR d_H1.27 (3H, s, 2-Me), 1.35 (3H,

s, 2-Me), 1.70–1.82 (2H, m, CH₂CHO), 2.62–2.78 (2H,

m, CH₂C@O), 3.45–3.47 (2H, m, CH₂OBn), 4.00 (1H,

ddd, J = 10.1, 6.2, 3.9 Hz, 4-H), 4.22 (1H, q,

J = 6.2 Hz, 5-H), 4.45 (1H, d, J = 11.9 Hz, CHPh),

4.49 (1H, d, J = 11.9 Hz, CHPh), 5.48 (0.35H, s,

CHCN), 7.21–7.35 (5H, m, Ph-H₅), 7.45 (1.3H, d,

J = 8.8 Hz, Ar 2,6-H₂), 7.55 (1.3H, d, J = 8.8 Hz, Ar

3,5-H₂), 7.59 (0.7H, d, J = 8.6 Hz, Ar 3,5-H₂), 7.76

(0.7H, d, J = 8.6 Hz, Ar 2,6-H₂), 9.35 (0.65H, s, OH);

MS m/z 474.1100 (M+H) (C₂₄H₂₇⁸¹BrNO₄requires

474.1102), 472.1094 (M+H) (C₂₄H₂₇⁷⁹BrNO₄requires

472.1123), 415/413 (MꢀC₂H₄NO), 91 (Bn).

6.29. (4R,5S)-5-Benzyloxymethyl-4-(-4-(4-chlorophenyl)-

4-cyano-3-methoxybut-3-enyl)-2,2-dimethyl-1,3-dioxo-

lane (24b) and 6-(2-((4R,5S)-5-benzyloxymethyl-2,2-

dimethyl-1,3-dioxolan-4-yl)ethyl)-5-(4-chlorophenyl)-

pyrimidine-2,4-diamine (25b)

6.27. (4R,5S)-5-Benzyloxymethyl-4-(4-(3,4-dichlorophe-

nyl)-4-cyano-3-oxobutyl)-2,2-dimethyl-1,3-dioxolane (22d)/

(4R,5S)-5-benzyloxymethyl-4-(4-cyano-3-hydroxy-4-(3,4-

dichlorophenyl)but-3-enyl)-2,2-dimethyl-1,3-dioxolane

(23d)

Compound 22b/23b was treated with CH₂N₂, as for the

synthesis of 24a, to give 24b (97%) as a pale yellow oil:

NMR d_H1.36 (3H, s, 2-Me), 1.45 (3H, s, 2-Me), 1.74

(1H, m, CHCHO), 1.85 (1H, m, CHCHO), 2.80 (1H,

m, CHC@C), 2.94 (1H, m, CHC@C), 3.51 (1H, dd,

J = 11.7, 6.0 Hz, CHOBn), 3.53 (1H, dd, J = 11.7,

6.0 Hz, CHOBn), 3.80 (3H, s, OMe), 4.22 (1 H, ddd,

J = 9.4, 6.0, 3.1 Hz, 4-H), 4.33 (1H, q, J = 6.0 Hz, 5-

H), 4.50 (1H, d, J = 12.1 Hz, CHPh), 4.59 (1H, d,

J = 12.1 Hz, CHPh), 7.28 (2H, d, J = 8.6 Hz, Ar 2,6-

H₂), 7.30–7.36 (5H, m, Ph-H₅), 7.52 (2H, d,

J = 8.6 Hz, Ar 3,5-H₂); MS m/z 444.1746 (M+H)

(C₂₅H₂₉³⁷ClNO₄requires 444.1755), 442.1764 (M+H)

(C₂₅H₂₉³⁵ClNO₄requires 442.1785), 428/426 (MꢀCH₃),

386/384 (MꢀC₂H₃NO), 91 (Bn). Compound 24b was

treated with guanidine, as for the synthesis of 25a (chro-

matographic eluant CHCl₃/MeOH, 9:1), to give 25b

(25%) as a pale buﬀ solid: mp 55–56 ꢁC; NMR d_H1.28

(6H, s, Me₂), 1.62–1.71 (2H, m, CH₂CHO), 2.25 (1H,

ddd, J = 13.5, 10.1, 5.9 Hz, Pyr-CH), 2.51 (1H, ddd,

J = 13.5, 10.1, 5.9 Hz, Pyr-CH), 3.43 (2H, d,

J = 5.9 Hz, CH₂OBn), 4.01 (1H, ddd, J = 10.1, 5.9,

3,4-Dichlorophenylacetonitrile and 21 were condensed,

as for the synthesis of 22a/23a, to give 22d/23d (16%)

as a highly hygroscopic white solid: NMR d_H1.41

(3H, s, 2-Me), 1.52 (3H, s, 2-Me), 1.73–1.87 (2H, m,

CH₂CHO), 2.71–2.84 (2H, m, CH₂C@O), 3.47 (1H,

dd, J = 11.7, 6.0 Hz, CHOBn), 3.49 (1H, dd, J = 11.7,

6.0 Hz, CHOBn), 3.98 (1H, m, 4-H), 4.22 (1H, q,

J = 6.0 Hz, 5-H), 4.47 (1H, d, J = 12.3 Hz, CHPh),

4.56 (1H, d, J = 12.3 Hz, CHPh), 7.26-7.37 (5H, m,

Ph-H₅), 7.45 (1H, d, J = 8.6 Hz, Ar 5-H), 7.50 (1H,

dd, J = 8.6, 2.0 Hz, Ar 6-H), 7.83 (1H, d, J = 2.0 Hz,

Ar 2-H), 9.61 (1H, s, OH); MS m/z 466.1178 (M+H)

(C₂₄H₂₆³⁷Cl₂NO₄requires 466.1179), 464.1193 (M+H)

(C₂₄H₂₆³⁷Cl³⁵ClNO₄requires 464.1209), 462.1217

(M+H) (C₂₄H₂₆³⁵Cl₂NO₄requires 462.1238), 407/405/

403 (MꢀC₂H₄NO), 91 (Bn).

4568

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4.3 Hz, dioxolane 4-H), 4.21 (1H, q, J = 5.9 Hz, dioxo-

lane 5-H), 4.47 (1H, d, J = 12.1 Hz, CHPh), 4.56 (1H,

d, J = 12.1 Hz, CHPh), 4.82 (2H, br, NH₂), 5.23 (2H,

br, NH₂), 7.14 (1H, d, J = 8.2 Hz, Ar 2-H), 7.15 (1H,

d, J = 8.2 Hz, Ar 6-H), 7.27–7.37 (5H, m, Ph-H₅), 7.39

(2H, d, J = 8.2 Hz, Ar 3,5-H₂); MS m/z 471.1992

(M+H) (C₂₅H₃₀³⁷ClN₄O₃requires 471.1976), 469.2005

(M+H) (C₂₅H₃₀³⁵ClN₄O₄requires 469.2006), 236/234

(MꢀC₁₄H₁₈O₃), 91 (Bn).

2.1 Hz, Ar 6-H), 7.73 (1H, d, J = 2.1 Hz, Ar 2-H); MS

m/z 480.1319 (M+H) (C₂₅H₂₈³⁷Cl₂NO₄requires

480.1336), 478.1344 (M+H) (C₂₅H₂₈³⁷Cl³⁵ClNO₄re-

quires 478.1365), 476.1367 (M+H) (C₂₅H₂₈³⁵Cl₂NO₄re-

quires 476.1395), 422/420/418 (MꢀC₂H₃NO), 91 (Bn).

Compound 24d was treated with guanidine, as for the

synthesis of 25a, to give 25d (47%) as a pale buﬀ solid:

mp 67–69 ꢁC; IR m_max3411, 1637 cm^ꢀ1; NMR d_H1.27

(6H, s, Me₂), 1.60 (1H, m, CHCHO), 1.73 (1H, m,

CHCHO), 2.25 (1 H, m, Pyr-CH), 2.47 (1H, m, Pyr-

CH), 3.44 (2H, d, J = 6.0 Hz, CH₂OBn), 3.99 (1H,

ddd, J = 9.8, 6.0, 3.5 Hz, dioxolane 4-H), 4.20 (1H, q,

J = 6.0 Hz, dioxolane 5-H), 4.47 (1H, d, J = 12.3 Hz,

CHPh), 4.56 (1H, d, J = 12.3 Hz, CHPh), 4.85 (2H, br,

NH₂), 5.21 (2H, br, NH₂), 7.03 (1H, dd, J = 8.0, 2.0 Hz,

Ar 6-H), 7.26-7.32 (5H, m, Ph-H₅), 7.35 (1H, d,

J = 2.0 Hz, Ar 2-H), 7.46 (1H, d, J = 8.0 Hz, Ar 5-H);

MS m/z 507.1563 (M+H) (C₂₅H₂₉³⁷Cl₂N₄O₃requires

507.1557), 505.1586 (M+H) (C₂₅H₂₉³⁷Cl³⁵ClN₄O₃

requires 505.1587), 503.1617 (M+H) (C₂₅H₂₉³⁵Cl₂N₄O₃

requires 503.1616), 91 (Bn).

6.30. (4R,5S)-5-Benzyloxymethyl-4-(4-(4-bromophenyl)-

4-cyano-3-methoxybut-3-enyl)-2,2-dimethyl-1,3-dioxolane

(24c) and 6-(2-((4R,5S)-5-benzyloxymethyl-2,2-dimethyl-

1,3-dioxolan-4-yl)ethyl)-5-(4-bromophenyl)pyrimidine-

2,4-diamine (25c)

Compound 22c/23c was treated with CH₂N₂, as for the

synthesis of 24a, to give 24c (87%) as a pale yellow oil:

NMR d_H1.36 (3H, s, 2-Me), 1.45 (3H, s, 2-Me), 1.75–

1.88 (2H, m, CH₂CHO), 2.80 (1H, m, CHC@C), 2.93

(1 H, m, CHC@C), 3.51 (1H, dd, J = 11.7, 5.9 Hz,

CH₂OBn), 3.53 (1 H, dd, J = 11.7, 5.9 Hz, CH₂OBn),

3.80 (3H, s, OMe), 4.21 (1 H, ddd, J = 9.4, 5.9, 3.1 Hz,

4-H), 4.32 (1H, q, J = 5.9 Hz, 5-H), 4.50 (1H, d,

J = 11.9 Hz, CHPh), 4.59 (1H, d, J = 11.9 Hz, CHPh),

7.27–7.32 (5H, m, Ph-H₅), 7.43 (2H, d, J = 8.6 Hz, Ar

2,6-H₂), 7.47 (2H, d, J = 9.0 Hz, Ar 3,5-H₂); MS m/z

488.1255 (M+H) (C₂₅H₂₉⁸¹BrNO₄requires 488.1259),

486.1263 (M+H) (C₂₅H₂₉⁷⁹BrNO₄requires 486.1279),

430/428 (MꢀC₂H₃NO), 91 (Bn). Compound 24c was

treated with guanidine, as for the synthesis of 25a, to

give 25c (57%) as a pale buﬀ solid: mp 62–64 ꢁC; IR m_max

3462, 1635 cm^ꢀ1; NMR d_H1.26 (6H, s, Me₂), 1.58–1.63

(2H, m, CH₂CHO), 2.22 (1H, ddd, J = 13.3, 10.1,

5.9 Hz, Pyr-CH), 2.49 (1H, ddd, J = 13.3, 10.1, 5.9 Hz,

Pyr-CH), 3.41 (2H, d, J = 5.9 Hz, CH₂OBn), 4.00 (1H,

ddd, J = 9.8, 5.9, 3.5 Hz, dioxolane 4-H), 4.20 (1H, q,

J = 5.9 Hz, dioxolane 5-H), 4.45 (1H, d, J = 12.1 Hz,

CHPh), 4.54 (1H, d, J = 12.1 Hz, CHPh), 4.65 (2H, br,

NH₂), 5.06 (2H, br, NH₂), 7.04 (1H, d, J = 7.8 Hz, Ar

2-H), 7.06 (1H, d, J = 8.2 Hz, Ar 6-H), 7.23–7.33 (5H,

m, Ph-H₅), 7.50 (2H, d, J = 8.2 Hz, Ar 3,5-H₂); MS m/

6.32. 6-((3R,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-

phenylpyrimidine-2,4-diamine (26a)

Compound 25a (700 mg, 1.6 mmol) was stirred for 16 h

with aq CF₃CO₂H (30%, 70 mL). Evaporation and

chromatography (CHCl₃/MeOH, 7:3) gave 26a

(600 mg, 95%) as a highly hygroscopic pale yellow solid:

NMR (CD₃OD) d_H1.69 (1H, m, 2⁰-H), 1.88 (1H, m, 2⁰-

H), 2.43 (1H, m, 1⁰-H), 2.60 (1H, ddd, J = 15.3, 10.6,

5.5 Hz, 1⁰-H), 3.43–3.47 (2H, m, 3⁰,4⁰-H₂), 3.49–3.56

(2H, m, 5⁰-H₂), 4.52 (1H, d, J = 12.6 Hz, CHPh), 4.56

(1H, d, J = 12.6 Hz, CHPh), 7.27–7.41 (6 H, m, 2· Ph

3,4,5-H₃), 7.47–7.58 (4H, m, 2· Ph 2,6-H₂); MS m/z

417 (M+Na), 395.2097 (M+H) (C₂₂H₂₇N₄O₃requires

395.2083), 243 (MꢀC₉H₁₁O₂), 213 (MꢀC₁₀H₁₃O₃), 91

(Bn).

6.33. 6-((3R,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-(3-

chlorophenyl)pyrimidine-2,4-diamine (26b)

z

515.1483 (M+H) (C₂₅H₃₀⁸¹BrN₄O₃requires

Compound 25b was treated with aq CF₃CO₂H, as for the

synthesis of 25a, to give 26b (87%) as a white solid: mp

131–133 ꢁC; NMR (CD₃OD) d_H1.64 (1H, m, 2⁰-H),

1.82 (1H, m, 2⁰-H), 2.37 (1H, m, 1⁰-H), 2.51 (1H, m, 1⁰-

H), 3.49–3.55 (2H, m, 3⁰,4⁰-H₂), 3.57-3.64 (2H, m, 5⁰-

H₂), 4.52 (1H, d, J = 12.5 Hz, CHPh), 4.56 (1H, d,

J = 12.5 Hz, CHPh), 7.24 (2H, d, J = 8.6 Hz, Ar 2,6-

H₂), 7.33–7.37 (5H, m, Ph-H₅), 7.47 (2H, d, J = 8.6 Hz,

Ar 3,5-H₂); NMR (CD₃OD) d_C29.02, 30.84, 71.03,

71.61, 72.09, 72.99, 107.42, 121.01, 127.36, 127.58,

128.01, 129.32, 132.16, 134.14, 138.10, 161.81, 162.16,

163.66; MS m/z 453/451 (M+Na), 431.1657 (M+H)

(C₂₂H₂₆³⁷ClN₄O₃requires 431.1663), 429.1680 (M+H)

(C₂₂H₂₆³⁵ClN₄O₄requires 429.1693), 279/277 (MꢀC₉H₁₁

O₂), 249/247 (MꢀC₁₀H₁₃O₃), 91 (Bn).

515.1480), 513.1497 (M+H) (C₂₅H₃₀⁷⁹BrN₄O₃requires

513.1501), 499/497 (MꢀCH₃), 91 (Bn).

6.31. (4R,5S)-5-Benzyloxymethyl-4-(4-(3,4-dichlorophe-

nyl)-4-cyano-3-methoxybut-3-enyl)-2,2-dimethyl-1,3-

dioxolane (24d) and 6-(2-((4R,5S)-5-benzyloxymethyl-

2,2-dimethyl-1,3-dioxolan-4-yl)ethyl)-5-(3,4-dichlorophe-

nyl)pyrimidine-2,4-diamine (25d)

Compound 22d/23d was treated with CH₂N₂, as for the

synthesis of 24a, to give 24d (97%) as a pale yellow oil:

NMR d_H1.36 (3H, s, 2-Me), 1.45 (3H, s, 2-Me), 1.77

(1H, m, CHCHO), 1.85 (1H, m, CHCHO), 2.81 (1H,

m, CHC@C), 2.94 (1H, m, CHC@C), 3.51 (1H, dd,

J = 11.3, 6.0 Hz, CHOBn), 3.53 (1H, dd, J = 11.3,

6.0 Hz, CHOBn), 3.84 (3H, s, OMe), 4.21 (1 H, ddd,

J = 9.4, 6.0, 3.1 Hz, 4-H), 4.32 (1H, q, J = 6.0 Hz, 5-

H), 4.49 (1H, d, J = 12.1 Hz, CHPh), 4.58 (1H, d,

J = 12.1 Hz, CHPh), 7.26–7.32 (5H, m, Ph-H₅), 7.37

(1H, d, J = 8.6 Hz, Ar 5-H), 7.42 (1H, dd, J = 8.6,

6.34. 6-((3R,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-(3-

bromophenyl)pyrimidine-2,4-diamine (26c)

Compound 25c was treated with aq CF₃CO₂H, as

for the synthesis of 25a, to give 26c (91%) as a highly

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4569

hygroscopic pale buﬀ solid: NMR d_H1.65–1.84 (2H, m,

2⁰-H₂), 2.35–2.55 (2H, m, 1⁰-H₂), 3.52–3.71 (4H, m,

3⁰,4⁰,5⁰-H₄), 4.49 (1H, d, J = 12.1 Hz, CHPh), 4.54

(1H, d, J = 12.1 Hz, CHPh), 4.71 (2H, br, NH₂), 5.09

(2H, br, NH₂), 7.05 (2H, d, J = 8.4 Hz, Ar 2,6-H₂),

7.24–7.34 (5H, m, Ph-H₅), 7.54 (2H, d, J = 8.4 Hz, Ar

3,5-H₂); MS m/z 497/495 (M+Na), 475.1184 (M + H)

(C₂₂H₂₆⁸¹BrN₄O₃requires 475.1167), 473.1179 (M+H)

pale yellow solid: mp 133–135 ꢁC; NMR d_H1.42 (3H,

s, Me), 1.52 (3H, s, Me), 2.35-2.52 (2H, m, CH₂CHO),

2.66–2.84 (2H, m, CH₂C@O), 3.63 (1H, dd, J = 12.7,

4.9 Hz, CHOH), 3.73 (1H, dd, J = 12.7, 4.9 Hz,

CHOH), 4.21–4.28 (2H, m, 4,5-H₂), 7.35 (2H, d,

J = 9.0 Hz, Ar 2,6-H₂), 7.44 (2H, d, J = 9.0 Hz, Ar

3,5-H₂); MS m/z 340 (M+H) C₁₇H₂₀³⁷ClNO₄, 338

(M+H) C₁₇H₂₀³⁵ClNO₄, 322/320 (MꢀOH), 308/306

(MꢀCH₃O).

(C₂₂H₂₆⁷⁹BrN₄O₄

requires

473.1188),

323/321

(MꢀC₉H₁₁O₂), 293/291 (MꢀC₁₀H₁₃O₃), 91 (Bn).

6.38. ((4R,5S)-4-(4-(4-Bromophenyl)-4-cyano-3-oxobu-

tyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (27c)/

(4R,5S)-4-(4-(4-bromophenyl)-4-cyano-3-hydroxybut-3-

enyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (28c)

6.35. 6-((3R,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-(3,4-

dichlorophenyl)pyrimidine-2,4-diamine (26d)

Compound 25d was treated with aq CF₃CO₂H, as for

the synthesis of 25a, to give 26d (74%) as a pale yellow

solid: mp 123–125 ꢁC; NMR (CD₃OD) d_H1.60 (1H,

m, 2⁰-H), 1.82 (1H, m, 2⁰-H), 2.31 (1H, ddd, J = 14.2,

9.0, 5.9 Hz, 1⁰-H), 2.47 (1H, ddd, J = 14.2, 9.0, 5.9 Hz,

1⁰-H), 3.44-3.51 (2H, m, 3⁰,4⁰-H₂), 3.54–3.59 (2H, m,

5⁰-H₂), 4.47 (1H, d, J = 14.1 Hz, CHPh), 4.52 (1H, d,

J = 14.1 Hz, CHPh), 7.15 (1H, dd, J = 8.2, 1.9 Hz, Ar

6-H), 7.22–7.33 (5H, m, Ph-H₅), 7.41 (1H, d, J = 1.9

Hz, Ar 2-H), 7.57 (1H, d, J = 8.2 Hz, Ar 5-H); NMR

(CD₃OD) d_C23.30, 30.97, 71.23, 71.58, 72.99, 106.22,

181.16, 127.31, 127.55, 127.99, 130.64, 131.15,

131.95, 132.61, 134.54, 138.17, 162.09, 162.83, 163.16;

MS m/z 489/487/485 (M+Na), 467.1258 (M+H)

(C₂₂H₂₅³⁷Cl₂N₄O₃requires 467.1244), 465.1272 (M+H)

(C₂₂H₂₅³⁷Cl³⁵ClN₄O₃requires 465.1274), 463.1293

(M+H) (C₂₂H₂₅³⁵Cl₂N₄O₃requires 463.1303), 315/313/

311 (MꢀC₉H₁₁O₂), 91 (Bn).

4-Bromophenylacetonitrile was condensed with 20, as

for the synthesis of 27a/28a, to give 27c/28c (6%) as a

pale yellow solid: mp 128–130 ꢁC; NMR d_H1.41 (3H,

s, Me), 1.51 (3H, s, Me), 2.39–2.48 (2H, m, CH₂CHO),

2.69–2.79 (2H, m, CH₂C@O), 3.62 (1H, dd, J = 11.6,

5.3 Hz, CHOH), 3.73 (1H, dd, J = 11.6, 5.3 Hz,

CHOH), 4.19–4.27 (2H, m, 4,5-H₂), 7.30 (2H, d,

J = 8.5 Hz, Ar 2,6-H₂), 7.47 (2H, d, J = 8.5 Hz, Ar 3,5-

H₂); MS m/z 382.0480 (M+H) (C₁₇H₂₀⁸¹BrNO₄requires

382.0476), 380.0475 (M+H) (C₁₇H₂₀⁷⁹BrNO₄requires

380.0497), 368/366 (MꢀCH₃), 342/340 (MꢀC₂H₃N),

326/324 (MꢀC₂H₃NO).

6.39. (4R,5S)-4-(4-Cyano-4-(3,4-dichlorophenyl)-3-oxobu-

tyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (27d)/

(4R,5S)-4-(4-cyano-4-(3,4-dichlorophenyl)-3-hydroxybut-

3-enyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (28d)

6.36. (4R,5S)-4-(4-Cyano-3-oxo-4-phenylbutyl)-5-hydroxy-

methyl-2,2-dimethyl-1,3-dioxolane (27a)/(4R,5S)-4-(4-

cyano-3-hydroxy-4-phenylbut-3-enyl)-5-hydroxymethyl-

2,2-dimethyl-1,3-dioxolane (28a)

3,4-Dichlorophenylacetonitrile was condensed with 20,

as for the synthesis of 27a/28a, to give 27d/28d (7%) as

a pale yellow solid: mp 117–118 ꢁC; IR m_max3424,

2209, 1718 cm^ꢀ1; NMR d_H1.42 (3H, s, Me), 1.52 (3H,

s, Me), 2.38–2.47 (2H, m, CH₂CHO), 2.72–2.84 (2H,

m, CH₂C@O), 3.64 (1H, dd, J = 11.5, 5.5 Hz, CHOH),

3.75 (1H, dd, J = 11.5, 5.5 Hz, CHOH), 4.18–4.29 (2H,

m, 4,5-H₂), 7.27 (1H, dd, J = 8.5, 2.2 Hz, Ar 6-H),

7.43 (1H, d, J = 8.5 Hz, Ar 5-H), 7.54 (1H, d,

J = 2.2 Hz, Ar 2-H). MS m/z 375/373/371 (M + H),

334/332/330 (MꢀC₂H₂N), 316/314/312 (MꢀC₂H₃NO).

LiN(SiMe₃)₂(1.0 M in THF, 30 mL, 30 mmol) was added

to phenylacetonitrile (1.75 g, 15 mmol) in dry Et₂O

(15 mL) under N₂at ꢀ78 ꢁC. After 10 min, 20 (3.5 g,

15 mmol) was added. The mixture was warmed to 20 ꢁC

and was stirred for 72 h. Water was added. The solution

was washed twice (Et₂O) before being acidiﬁed to pH 6

with aq citric acid (1 M) in the presence of EtOAc. The

EtOAc phase was separated and washed with water. Dry-

ing, evaporation and chromatography (EtOAc/hexane,

3:1) gave 27a/28a (450 mg, 10%) as a pale yellow oil:

NMR d_H1.30 (3H, s, Me), 1.40 (3H, s, Me), 1.74–1.81

(2H, m, CH₂CHO), 2.71 (1H, m, CHC@O), 2.83 (1H,

m, CHC@O), 3.62 (2H, d, J = 5.5 Hz, CH₂OH), 4.07

(1H, m, 4-H), 4.15 (1H, m, 5-H), 4.79 (1H, s, CHCN),

7.36–7.50 (5H, m, Ph-H₅); MS m/z 303.1464 (M+H)

(C₁₇H₂₁NO₄requires 303.1470), 287 (MꢀCH₃), 271

(MꢀCH₃O), 245 (MꢀC₂H₃NO).

6.40. (4R,5S)-4-(4-Cyano-3-methoxy-4-phenylbut-3-enyl)-

2,2-dimethyl-5-hydroxymethyl-1,3-dioxolane (29a) and

6-(2-((4R,5S)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxo-

lan-4-yl)ethyl)-5-phenylpyrimidine-2,4-diamine (30a)

Compound 27a/28a was treated with CH₂N₂, as for the

synthesis of 24a, to give 29a (330 mg, 95%) as a pale yel-

low oil: NMR d_H1.40 (3H, s, 2-Me), 1.51 (3H, s, 2-Me),

1.87–2.01 (2H, m, CH₂CHO), 2.84–3.02 (2H, m,

CH₂C@C), 3.69 (2H, d, J = 5.9 Hz, CH₂OH), 3.86

(3H, s, OMe), 4.22–4.27 (2H, m, 4,5-H₂), 7.24–7.42

(5H, m, Ph-H₅); MS m/z 318.1707 (M+H) (C₁₈H₂₄NO₄

requires 318.1705), 302 (MꢀMe), 277 (MꢀC₂H₂N),

258 (MꢀC₂H₅NO). Compound 29a was treated with

guanidine, as for the synthesis of 25a (reaction time 4

h, chromatographic eluant CHCl₃/MeOH, 9:1), to give

30a (42%) as a pale buﬀ solid: mp 72–75 ꢁC; NMR

(D₂O) d_H1.27 (3H, s, Me), 1.31 (3H, s, Me), 1.75–1.88

6.37. (4R,5S)-4-(4-(4-Chlorophenyl)-4-cyano-3-oxobutyl)-

5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (27b)/(4R,5S)-

4-(4-(4-chlorophenyl)-4-cyano-3-hydroxybut-3-enyl)-5-

hydroxymethyl-2,2-dimethyl-1,3-dioxolane (28b)

4-Chlorophenylacetonitrile was condensed with 20, as

for the synthesis of 27a/28a, to give 27b/28b (7%) as a

4570

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

(2H, m, CH₂CHO), 2.32 (1H, ddd, J = 15.9, 9.7, 6.0 Hz,

Pyr-CH), 2.49 (1H, ddd, J = 15.9, 8.7, 7.4 Hz, Pyr-CH),

3.53 (1H, dd, J = 11.6, 5.9 Hz, CHOH), 3.65 (1H, dd,

J = 11.6, 5.9 Hz, CHOH), 4.05 (1H, dd, J = 12.2,

5.9 Hz, dioxolane 4-H), 4.12 (1H, q, J = 5.9 Hz, dioxo-

lane 5-H), 4.63 (2H, br, NH₂), 4.95 (2H, br, NH₂),

7.21 (1H, d, J = 7.9 Hz, Ar 2-H), 7.22 (1H, d,

J = 7.9 Hz, Ar 6-H), 7.36 (1H, t, J = 7.9 Hz, Ar 4-H),

7.21 (2H, t, J = 7.9 Hz, Ar 3,5-H₂); NMR (D₂O) d_C

23.01, 25.58, 25.70, 28.17, 58.60, 74.07, 75.63, 105.27,

106.26, 125.50, 126.82, 126.85, 127.94, 158.71, 159.82,

162.92; MS m/z 345.1935 (M+H) (C₁₉H₂₄N₄O₃requires

345.1926), 329 (MꢀMe), 200 (MꢀC₇H₁₂O₃).

dioxolane 4-H), 4.08 (1H, q, J = 5.9 Hz, dioxolane 5-

H), 7.21 (2H, d, J = 7.7 Hz, Ar 2,6-H₂), 7.66 (2H, d,

J = 7.7 Hz, Ar 3,5-H₂); NMR (CD₃OD) d_C24.43,

27.11, 28.49, 30.66, 60.32, 76.63, 77.89, 107.20, 107.82,

121.84, 132.21, 132.57, 133.50, 160.82, 162.98, 163.71;

MS m/z 425.1007 (M+H) (C₁₈H₂₄⁸¹BrN₄O₃requires

425.1011), 423.1019 (M+H) (C₁₈H₂₄⁷⁹BrN₄O₄requires

423.1013), 280/278 (MꢀC₇H₁₂O₃), 186 (MꢀC₈H₁₃BrO₃).

6.43. (4R,5S)-4-(4-(3,4-Dichlorophenyl)-4-cyano-3-meth-

oxybut-3-enyl)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxo-

lane (29d) and 5-(3,4-dichlorophenyl)-6-(2-((4R,5S)-5-

hydroxymethyl-2,2-dimethyl-1,3-dioxolan-4-yl)ethyl)pyrim-

idine-2,4-diamine (30d)

6.41. (4R,5S)-4-(4-(4-Chlorophenyl)-4-cyano-3-methoxy-

but-3-enyl)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxolane

(29b) and 5-(4-chlorophenyl)-6-(2-((4R,5S)-2,2-dimethyl-

5-hydroxymethyl-1,3-dioxolan-4-yl)ethyl)pyrimidine-2,4-

diamine (30b)

Compound 27c/28c was treated with CH₂N₂, as for the

synthesis of 24a, to give 29d (91%) as a pale yellow oil:

IR m_max3467, 2210, 1597 cm^ꢀ1; NMR d_H1.40 (3H, s, 2-

Me), 1.50 (3H, s, 2-Me), 2.30–2.40 (2H, m, CH₂CHO),

2.72–2.80 (2H, m, CH₂C@C), 3.37 (3H, s, OMe), 3.58–

3.74 (2H, m, CH₂OH), 4.18–4.26 (2H, m, 4,5-H₂), 7.51

(1H, d, J = 8.3 Hz, Ar 5-H), 7.84 (1H, dd, J = 8.3,

2.0 Hz, Ar 6-H), 8.10 (1H, d, J = 2.0 Hz, Ar 2-H). Com-

pound 29d was treated with guanidine, as for the synthe-

sis of 25a (reaction time 6 h, chromatographic eluant

CHCl₃/MeOH, 9:1), to give 30d (9%) as a white solid:

mp 114–115 ꢁC; NMR d_H1.29 (3H, s, Me), 1.33 (3H,

s, Me), 1.78 (1H, m, CHCHO), 1.88 (1H, m, CHCHO),

2.34 (1H, m, Pyr-CH), 2.48 (1 H, m, Pyr-CH), 3.58 (1H,

dd, J = 11.4, 5.9 Hz, CHOH), 3.67 (1H, dd, J = 11.4,

5.9 Hz, CHOH), 4.05 (1H, m, dioxolane 4-H), 4.15

(1H, q, J = 5.6 Hz, dioxolane 5-H), 4.95 (2H, br,

NH₂), 5.53 (2H, br, NH₂), 7.08 (1H, dd, J = 8.4,

1.7 Hz, Ar 6-H), 7.38 (1H, d, J = 1.7 Hz, Ar 2-H), 7.52

(1H, d, J = 8.4 Hz, Ar 5-H); NMR d_C25.53, 28.04,

28.12, 30.35, 61.05, 65.83, 70.51, 106.62, 108.04, 130.15,

131.41, 131.45, 133.51, 134.4, 160.93, 162.22, 168.33;

MS m/z 417.1091 (M+H) (C₁₈H₂₃³⁷Cl₂N₄O₃requires

417.1088), 415.1103 (M+H) (C₁₈H₂₃³⁷Cl³⁵ClN₄O₃

requires 415.1117), 413.1129 (M+H) (C₁₈H₂₃³⁵Cl₂N₄O₃

requires 413.1147), 272/270/268 (MꢀC₇H₁₂O₃) 186

(MꢀC₈H₁₂Cl₂O₃).

Compound 27b/28b was treated with CH₂N₂, as for the

synthesis of 24a, to give 29b (90%) as a pale yellow oil.

Compound 29b was treated with guanidine, as for the

synthesis of 25a (reaction time 10 h, chromatographic elu-

ant CHCl₃/MeOH, 9:1), to give 30b (12%) as a white solid:

mp 94–95 ꢁC; NMR d_H1.27 (3H, s, Me), 1.30 (3H, s, Me)

1.73 (1H, m, CHCHO), 1.84 (1H, m, CHCHO), 2.29 (1H,

ddd, J = 13.4, 10.8, 5.1 Hz, Pyr-CH), 2.46 (1H, ddd,

J = 13.4, 10.4, 5.9 Hz, Pyr-CH), 3.56 (1H, dd, J = 11.7,

6.1 Hz, CHOH), 3.66 (1H, dd, J = 11.7, 6.1 Hz, CHOH),

4.02 (1H, dt, J = 8.1, 5.8 Hz, dioxolane 4-H), 4.13 (1H, q,

J = 5.8 Hz, dioxolane 5-H), 5.04 (2H, br, NH₂), 5.76 (2H,

br, NH₂), 7.16 (2H, d, J = 7.0 Hz, Ar 2,6-H₂), 7.42 (2H, d,

J = 7.0 Hz, Ar 3,5-H₂); NMR d_C25.57, 28.04, 28.42,

30.46, 60.90, 76.77, 77.09, 107.39, 107.94, 129.71,

132.64, 134.29, 160.82, 162.48, 164.40; MS m/z 381.1506

(M+H) (C₁₈H₂₄³⁷ClN₄O₃requires 381.1507), 379.1525

(M+H) (C₁₈H₂₄³⁵ClN₄O₄requires 379.1536), 236/234

(MꢀC₇H₁₂O₃), 188/186 (MꢀC₈H₁₃ClO₃).

6.42. (4R,5S)-4-(4-(4-Bromophenyl)-4-cyano-3-methoxy-

but-3-enyl)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxolane

(29c) and 5-(4-bromophenyl)-6-(2-((4R,5S)-2,2-dimethyl-

5-hydroxymethyl-1,3-dioxolan-4-yl)ethyl)pyrimidine-2,4-

diamine (30c)

6.44. Diethyl (R,R)-2,2-diethyl-1,3-dioxolane-4,5-

dicarboxylate (32)

Compound 27c/28c was treated with CH₂N₂, as for the

synthesis of 24a, to give 29c (90%) as a pale yellow oil:

IR m_max3435, 2243, 1592 cm^ꢀ1; NMR d_H1.42 (3H, s,

Me), 1.52 (3H, s, Me), 2.38–2.49 (2H, m, CH₂CHO),

2.74-2.80 (2H, m, CH₂C@C), 3.33 (3H, s, OMe), 3.64

(1H, dd, J = 11.1, 5.0 Hz, CHOH), 3.74 (1H, dd,

J = 11.1, 5.0 Hz, CHOH), 4.21–4.27 (2H, m, dioxolane

4,5-H₂), 7.29 (2H, d, J = 8.5 Hz, Ar 2,6-H₂), 7.50 (2 H,

d, J = 8.5 Hz, Ar 3,5-H₂). Compound 29c was treated

with guanidine, as for the synthesis of 25a (reaction time

10 h, chromatographic eluant CHCl₃/MeOH, 9:1), to

give 30c (20%) as a white solid: mp 124–125 ꢁC; NMR

d_H1.27 (6H, s, Me₂), 1.61–1.79 (2H, m, CH₂CHO),

2.26 (1H, ddd, J = 13.0, 10.6, 5.8 Hz, Pyr-CH), 2.46

(1H, ddd, J = 13.0, 10.6, 5.8 Hz, Pyr-CH), 3.50 (1H, dd,

J = 11.1, 5.9 Hz, CHOH), 3.54 (1H, dd, J = 11.1,

5.9 Hz, CHOH), 4.01 (1H, ddd, J = 9.9, 5.9, 3.9 Hz,

Diethyl (R,R)-2,3-dihydroxybutanedioate 31 (15.0 g,

70 mmol), 2,2-dimethoxypropane (8.0 g, 80 mmol) and

4-methylbenzenesulfonic acid (66 mg, 0.34 mmol) in

dichloromethane (200 mL) were heated under reﬂux

˚

through activated 4 A molecular sieves (33 g) in a Soxh-

let apparatus for 3 h. Na₂CO₃(83 mg, 1.0 mmol) was

added. Filtration, drying and evaporation gave 32

(16.0 g, 89%) as a pale buﬀ oil (lit.⁴⁷oil): NMR d_H1.32

(6H, t, J = 7.2 Hz, 2· CH₂CH₃), 1.50 (6H, s, CMe₂),

4.28 (4H, q, J = 7.2 Hz, 2· CH₂), 4.77 (2H, s, 4,5-H₂).

6.45. (S,S)-4,5-Di(hydroxymethyl)-2,2-dimethyl-1,3-

dioxolane (33)

LiAlH₄(6.0 g, 150 mmol) was heated in dry THF

(60 mL) for 30 min. Compound 32 (18.0 g, 70 mmol)

in dry THF (80 mL) was added during 1.5 h. The

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4571

mixture was heated under reﬂux for 5 h, then cooled to

0 ꢁC. Water (10 mL), aq NaOH (4 M, 10 mL) and water

(30 mL) were added. Filtration and evaporation gave

33. The solid was extracted with hot 1,4-dioxane; evap-

oration gave further 33 (total 7.0 g, 60%) as a pale yel-

low oil (lit.⁴⁸oil): NMR d_H1.41 (6H, s, Me₂), 2.65

(2H, br, 2· OH), 3.68–3.78 (4H, m, 2· CH₂), 3.97

(2H, m, 4,5-H₂).

matography (hexane / Et₂O 5:1) gave 37Z (800 g, 31%)

as a colourless oil (lit.⁵²oil): NMR d_H1.25 (3H, t,

J = 7.1 Hz, CH₂CH₃), 1.45 (6H, s, CMe₂), 3.68 (2H, d,

J = 3.1 Hz, CH₂OBn), 3.97 (1H, m, 4-H), 4.12 (2H, q,

J = 7.1 Hz, , CH₂Me), 4.56 (1 H, d, J = 12.1 Hz, CHPh),

4.62 (1H, d, J = 12.1 Hz, CHPh), 5.38 (1 H, td, J = 8.3,

1.2 Hz, 5-H), 5.92 (1H, dd, J = 11.7, 1.2 Hz, CHCO₂),

6.18 (1H, dd, J = 11.7, 8.3 Hz, CHC@CCO₂), 7.32-

7.37 (5H, m, Ph-H₅). Further elution gave 37E

(800 mg, 31%) as a colourless oil (lit.⁵³oil): NMR d_H

1.29 (3H, t, J = 7.0 Hz, CH₂CH₃), 1.43 (3H, s, 2-Me),

1.45 (3H, s, 2-Me), 3.62 (2H, d, J = 4.7 Hz, CH₂OBn),

3.95 (1H, dt, J = 8.6, 4.7 Hz, 4-H), 4.19 (2H, q,

J = 7.0 Hz, CH₂Me), 4.42 (1H, ddd, J = 8.6, 5.5,

1.4 Hz, 5-H), 4.56 (1H, J = 12.1 Hz, CHPh), 4.61 (1H,

d, J = 12.1 Hz, CHPh), 6.09 (1H, dd, J = 15.6, 1.4 Hz,

CHCO₂), 6.88 (1H, dd, J = 15.6, 5.5 Hz, CHC@CCO₂),

7.27-7.36 (5H, m, Ph-H₅).

6.46. (S,S)-4,5-Di(benzyloxymethyl)-2,2-dimethyl-1,3-

dioxolane (34) and (S,S)-4-benzyloxymethyl-5-hydroxy-

methyl-2,2-dimethyl-1,3-dioxolane (35)

NaH (60% oil, 1.4 g, 34 mmol) was stirred in dry DMF

(20 mL) under N₂for 30 min. Compound 33 (5.0 g,

31 mmol) in DMF (20 mL) was added dropwise and

the mixture was stirred for 30 min before BnCl (4.0 g,

32 mmol) was added. The mixture was stirred for 1.5 h,

then poured into ice-water (250 mL) and extracted thrice

with Et₂O. The combined extracts were washed with

water and brine. Drying, evaporation and chromatogra-

phy (hexane / Et₂O 1:1) gave 34 (2.2 g, 28%) as a pale yel-

low oil (lit.⁴⁹oil): NMR d_H1.42 (6H, s, Me₂), 3.54-3.66

(4H, m, 2· CH₂OBn), 4.02 (2H, m, 4,5-H₂), 4.54 (2H,

d, J = 12.3 Hz, 2· CHPh), 4.58 (2H, d, J = 12.3 Hz, 2·

6.49. Ethyl (4S,5S)-4-benzyloxymethyl-2,2-dimethyl-1,3-

dioxolane-5-propanoate (38)

A mixture of 37Z and 37E (620 mg, 1.9 mmol) was stir-

red in EtOH (25 mL) with Pd/C (5%, 30 mg) under H₂

for 1 h. Filtration (Celite^ꢂ), evaporation and chroma-

CHPh), 7.35 (10 H, m, 2· Ph-H₅). Further elution gave

tography (hexane/Et₂O, 4:1) gave 38 (400 mg, 63%) as

20

35 (3.2 g, 64%) as a pale yellow oil. ½aꢁ ¼ þ8:0^ꢂ(c 3.2,

20

D

a pale yellow oil: ½aꢁ ¼ ꢀ15^ꢂ(c 4.0, CHCl₃); NMR

D

23

D

CHCl₃) (lit.⁵⁰½aꢁ ¼ þ8:2^ꢂ(c 1.0, CHCl₃)); NMR d_H

d_H1.23 (3H, t, J = 7.0 Hz, CH₂CH₃), 1.38 (3H, s, 2-

Me), 1.39 (3H, s, 2-Me), 1.84 (1H, m, CHCH₂CO₂),

1.96 (1H, m, CHCH₂CO₂), 2.37–2.54 (2H, m, CH₂CO₂),

3.53–3.60 (2H, m, CH₂OBn), 3.80–3.87 (2H, m, 4,5-H₂),

4.12 (2H, q, J = 7.0 Hz, CH₂Me), 4.56 (1H, d,

J = 12.3 Hz, CHPh), 4.59 (1H, d, J = 12.3 Hz, CHPh),

7.32-7.34 (5H, m, Ph-H₅); MS m/z 323.1856 (M+H)

(C₁₉H₂₆O₅requires 323.1858), 265 (MꢀC₃H₅O), 91

(Bn).

1.41 (3H, s, Me), 1.42 (3H, s, Me), 2.33 (1H, dd,

J = 8.6, 4.3 Hz, OH), 3.55 (1H, dd, J = 9.8, 4.3 Hz,

CHOBn), 3.64-3.70 (2H, m, CHOH + CHOBn), 3.75

(1H, dt, J = 11.7, 4.3 Hz, CHOH), 3.94 (1H, dt,

J = 8.3, 4.3 Hz, 5-H), 4.05 (1H, dt, J = 8.3, 4.3 Hz,

4-H), 4.58 (2H, s, CH₂Ph), 7.29–7.35 (5H, m, Ph-H₅).

6.47. (4S,5R)-4-Benzyloxymethyl-2,2-dimethyl-1,3-dioxo-

lane-5-carboxaldehyde (4-O-benzyl-2,3-O-isopropylid-

ene-^L-threose) (36)

6.50. (4S,5S)-5-Benzyloxymethyl-4-(4-cyano-3-oxo-4-

phenylbutyl)-2,2-dimethyl-1,3-dioxolane (39a)/(4S,5S)-5-

benzyloxymethyl-4-(4-cyano-3-hydroxy-4-phenylbut-3-

enyl)-2,2-dimethyl-1,3-dioxolane (40a)

Compound 35 (3.6 g, 14 mmol) was stirred with pyridini-

um chlorochromate (3.6 g, 35 mmol), NaOAc (300 mg,

˚

3.5 mmol) and powdered 4 A molecular sieves (3.0 g) in

CH₂Cl₂(215 mL) under N₂for 3 h. The mixture was

passed through a bed of silica. The silica was extracted

with Et₂O. Evaporation of the solvent from the

Phenylacetonitrile was condensed with 38, as for the

synthesis of 22a/23a, to give 39a/40a (14%) as a pale yel-

low solid: mp 75–77 ꢁC; IR m_max2206, 1731 cm^ꢀ1; NMR

d_H1.39 (3H, s, Me), 1.41 (3H, s, Me), 1.86 (1H, m,

CHCHO), 2.00 (1H, m, CHCHO), 2.48–2.65 (2H, m,

CH₂C@O), 3.54–3.63 (2H, m, 4,5-H₂), 3.87–3.89 (2H,

m, CH₂OBn), 4.57 (1H, d, J = 12.5 Hz, CHPh), 4.61

(1H, d, J = 12.5 Hz, CHPh), 5.59 (1H, s, CHCN),

7.33–7.64 (8 H, m, Ph⁰3,4,5-H₃+ Ph-H₅), 8.11 (2H, d,

J = 7.0 Hz, Ph 2,6-H₂); MS m/z 392.1859 (MꢀH)

(C₂₄H₂₆NO₄requires 392.1861), 335 (MꢀC₂H₄NO),

317 (MꢀC₇H₆), 91 (Bn).

combined ﬁltrate and extract gave 36 (3.3 g, 93%) as a

20

D

pale yellow oil: ½aꢁ ¼ þ14^ꢂ(c 3, CHCl₃) (lit.⁵¹

20

D

½aꢁ ¼ þ16:2^ꢂ(c 1, CHCl₃); NMR d_H1.43 (3H, s, Me),

1.50 (3H, s, Me), 3.67 (2H, d, J = 4.0 Hz, CH₂OBn),

4.19–4.29 (2H, m, 4,5-H₂), 4.58 (1H, d, J = 10.5 Hz,

CHPh), 4.61 (1H, d, J = 10.5 Hz, CHPh), 7.25-7.36

(5H, m, Ph-H₅), 9.76 (1H, d, J = 1.5 Hz, CHO).

6.48. Ethyl (Z,4S,5S)-4-benzyloxymethyl-2,2-dimethyl-

1,3-dioxolane-5-propenoate (37Z) and ethyl (E,4S,5S)-4-

benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-5-propeno-

ate (37E)

6.51. (4S,5S)-5-Benzyloxymethyl-4-(4-(4-chlorophenyl)-

4-cyano-3-oxobutyl)-2,2-dimethyl-1,3-dioxolane (39b)/

(4S,5S)-5-benzyloxymethyl-4-(4-(4-chlorophenyl)-4-cya-

no-3-hydroxybut-3-enyl)-2,2-dimethyl-1,3-dioxolane (40b)

Compound 36 (2.0 g, 8.0 mmol), ethyl triphenylphos-

phoranylideneacetate (4.2 g, 16 mmol) and benzoic acid

(50 mg, 0.4 mmol) were heated at reﬂux in PhMe

(200 mL) under N₂for 4 h. The evaporation residue

was extracted thrice with Et₂O. Evaporation and chro-

4-Chlorophenylacetonitrile was condensed with 38, as

for the synthesis of 22a/23a (chromatographic eluant

EtOAc/hexane, 1:1), to give 39b/40b (41%) as a yellow

4572

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

oil: IR m_max2209, 1731 cm^ꢀ1; NMR d_H1.39 (3 H, s,

Me), 1.40 (3H, s, Me), 1.85 (1H, m, CHCHO), 2.00

(1H, m, CHCHO), 2.46–2.63 (2H, m, CH₂C@O),

3.53–3.62 (2H, m, 4,5-H₂), 3.83–3.87 (2H, m, CH₂OBn),

4.57 (1H, d, J = 12.1 Hz, CHPh), 4.60 (1H, d,

J = 12.1 Hz, CHPh), 7.26–7.40 (5H, m, Ph-H₅), 7.44

(2H, d, J = 8.6 Hz, Ar 3,5-H₂), 8.02 (2 H, d,

J = 8.6 Hz, Ar 2,6-H₂); MS m/z 430.1608 (M+H)

(C₂₄H₂₇³⁷ClNO₄requires 430.1599), 428.1623 (M+H)

6.54. (4S,5S)-5-Benzyloxymethyl-4-(-4-(4-chlorophenyl)-

4-cyano-3-methoxybut-3-enyl)-2,2-dimethyl-1,3-dioxo-

lane (41b) and 6-(2-((4S,5S)-5-benzyloxymethyl-2,2-

dimethyl-1,3-dioxolan-4yl)ethyl)-5-(4-chlorophenyl)pyrim-

idine-2,4-diamine (42b)

Compound 39b/40b was treated with CH₂N₂, as for

the synthesis of 24a, to give41b (91%) as a pale yel-

low oil: NMR d_H1.39 (3H, s, Me), 1.41 (3H, s,

Me), 1.62–1.75 (2H, m, CH₂CHO), 2.86-2.96 (2H,

m, CH₂C@C), 3.52–3.65 (2H, m, 4,5-H₂), 3.81 (3H,

s, OMe), 3.84–3.93 (2H, m, CH₂OBn), 4.54 (1H, d,

J = 11.1 Hz, CHPh), 4.58 (1H, d, J = 11.1 Hz, CHPh),

7.24–7.38 (9H, m, Ph-H₅+Ar-H₄). Compound 41b was

condensed with guanidine, as for the synthesis of 25a

(reaction time 4 h, chromatographic eluant CH₂Cl₂/

MeOH, 4:1), to give 42b (48%) as a highly hygro-

scopic pale yellow solid: IR m_max3475, 3414,

1618 cm^ꢀ1; NMR d_H1.28 (3H, s, Me), 1.31 (3H, s,

Me), 1.73 (1H, m, CHCHO), 1.83 (1H, m, CHCHO),

2.30 (1H, ddd, J = 13.5, 10.5, 5.7 Hz, Pyr-CH), 2.45

(1H, ddd, J = 13.5, 10.5, 5.7 Hz, Pyr-CH), 3.41–3.49

(2H, m, CH₂OBn), 3.66 (1H, dt, J = 8.2, 3.5 Hz,

dioxolane 4-H), 3.51 (1H, m, dioxolane 5-H), 4.50

(1 H, d, J = 12.1 Hz, CHPh), 4.54 (1H, d,

J = 12.1 Hz, CHPh), 4.69 (2 H, br, NH₂), 5.11 (2H,

br, NH₂), 7.10–7.35 (9 H, m, Ph-H₅+Ar-H₄); MSm/z

(C₂₄H₂₇³⁵ClNO₄

(MꢀC₂H₃NO), 91 (Bn).

requires

428.1628),

372/370

6.52. (4S,5S)-5-Benzyloxymethyl-4-(4-(4-bromophenyl)-

4-cyano-3-oxobutyl)-2,2-dimethyl-1,3-dioxolane (39c)/

(4S,5S)-5-benzyloxymethyl-4-(4-(4-bromophenyl)-4-cya-

no-3-hydroxybut-3-enyl)-2,2-dimethyl-1,3-dioxolane

(39c)

4-Bromophenylacetonitrile was condensed with 38, as

for the synthesis of 22a/23a, to give 39c/40c (24%): as

a yellow oil: IR m_max2208, 1718 cm^ꢀ1; NMR d_H1.33

(3H, s, Me), 1.34 (3H, s, Me), 1.75 (1H, m, CHCHO),

1.91 (1H, m, CHCHO), 2.69 (1H, m, CHC@O), 2.79

(1H, m, CHC@O), 3.55–3.65 (2H, m, CH₂OBn), 3.93

(1H, m, 5-H), 4.03 (1H, dt, J = 8.0, 3.7 Hz, 4-H), 4.53

(1H, d, J = 12.0 Hz, CHPh), 4.61 (1H, d, J = 12.0 Hz,

CHPh), 7.22 (2H, d, J = 8.1 Hz, Ar 2,6-H₂), 7.26–7.36

(5H, m, Ph-H₅), 7.53 (2H, d, J = 8.8 Hz, Ar 3,5-H₂);

MS m/z 474.1103 (M+H) (C₂₄H₂₇⁸¹BrNO₄requires

474.1102), 472.1103 (M+H) (C₂₄H₂₇⁷⁹BrNO₄requires

472.1123), 415/413 (MꢀC₂H₄NO), 91 (Bn).

471.1979

(M+H)

(C₂₅H₃₀³⁷ClN₄O₃

requires

471.1976), 469.1999 (M+H) (C₂₅H₃₀³⁵ClN₄O₃requires

469.2006), 363/361 (MꢀC₇H₇O), 91 (Bn).

6.55. (4S,5S)-5-Benzyloxymethyl-4-(-4-(4-bromophenyl)-

4-cyano-3-methoxybut-3-enyl)-2,2-dimethyl-1,3-dioxo-

lane (41c) and 6-(2-((4S,5S)-5-benzyloxymethyl-2,2-

dimethyl-1,3-dioxolan-4yl)-ethyl)-5-(4-bromophenyl)pyrim-

idine-2,4-diamine (42c)

6.53. (4S,5S)-5-Benzyloxymethyl-4-(4-cyano-3-methoxy-

4-phenylbut-3-enyl)-2,2-dimethyl-1,3-dioxolane (41a) and

6-(2-((4S,5S)-5-benzyloxymethyl-2,2-dimethyl-1,3-dioxo-

lan-4-yl)ethyl)-5-phenylpyrimidine-2,4-diamine (42a)

Compound 39c/40c was treated with CH₂N₂, as for the

synthesis of 24a, to give41c (93%) as a pale yellow oil:

NMR d_H1.39 (3H, s, Me), 1.41 (3H, s, Me), 1.78–1.86

(2H, m, CH₂CHO), 2.85–2.95 (2H, m, CH₂C@C),

3.52–3.68 (2H, m, 4,5-H₂), 3.81 (3H, s, OMe), 3.84–

3.94 (2H, m, CH₂OBn), 4.54 (1H, d, J = 12.1 Hz,

CHPh), 4.59 (1H, d, J = 12.1 Hz, CHPh), 7.22-7.42

(9 H, m, Ph-H₅+Ar-H₄); MS m/z 488.1255 (M+H)

(C₂₅H₂₉⁸¹BrNO₄requires 488.1259), 486.1258 (M+H)

(C₂₅H₂₉⁷⁹BrNO₄requires 486.1279), 91 (Bn). Com-

pound 41c was condensed with guanidine, as for the

synthesis of 42a, to give 42c (53%) as a highly hygro-

scopic buﬀ solid: NMR d_H1.31 (3H, s, Me), 1.34 (3H,

s, Me), 1.75 (1H, m, CHCHO), 1.88 (1H, m,

CHCHO), 2.33 (1H, ddd, J = 13.7, 10.5, 5.9 Hz, Pyr-

CH), 2.48 (1H, ddd, J = 13.7, 10.5, 5.9 Hz, Pyr-CH),

3.45–3.53 (2H, m, CH₂OBn), 3.70 (1H, dt, J = 7.8,

3.5 Hz, dioxolane 4-H), 3.77 (1H, m, dioxolane 5-H),

4.51 (1 H, d, J = 12.1 Hz, CHPh), 4.57 (1H, d,

J = 12.1 Hz, CHPh), 4.76 (2 H, br, NH₂), 5.18 (2H,

br, NH₂), 7.09 (2H, d, J = 8.6 Hz, Ar 2,6-H₂), 7.28–

7.38 (5H, m, Ph-H₅), 7.54 (2H, d, J = 8.6 Hz, Ar 3,5-

H₂); MS m/z 515.1488 (M+H) (C₂₅H₃₀⁸¹BrN₄O₃re-

quires 515.1480), 513.1500 (M+H) (C₂₅H₃₀⁷⁹BrN₄O₄

requires 513.1501), 487/485 (MꢀC₂H₅), 407/405

(MꢀC₇H₇O), 91 (Bn).

Compound 39a/40a was treated with CH₂N₂, as for the

synthesis of 24a, to give41a (79%) as a pale yellow oil:

IR m_max2208, 1605 cm^ꢀ1; NMR d_H1.45 (3H, s, Me),

1.49 (3H, s, Me), 1.83 (1H, m, CHCHO), 1.98 (1H,

m, CHCHO), 2.40–2.59 (2H, m, CH₂C@C), 3.55–3.60

(2H, m, 4,5-H₂), 3.75 (3H, s, OMe), 3.81–3.85 (2H,

m, CH₂OBn), 4.55 (1H, d, J = 12.7 Hz, CHPh), 4.58

(1H, d, J = 12.7 Hz, CHPh), 7.14–7.45 (10 H, m, 2·

Ph-H₅); MS m/z 408.2184 (M+H) (C₂₅H₃₀NO₄requires

408.2174), 391 (MꢀCH₄), 380 (MꢀHCN), 91 (Bn).

Compound 41a was condensed with guanidine, as for

the synthesis of 25a (reaction time 4 h, chromato-

graphic eluant CHCl₃/MeOH, 9:1), to give 42a (67%)

as a highly hygroscopic pale yellow solid: IR m_max

3454, 1664 cm^ꢀ1; NMR d_H1.26 (3H, s, Me), 1.29

(3H, s, Me), 1.71–1.91 (2H, m, CH₂CHO), 2.33 (1H,

ddd, J = 13.5, 10.3, 5.8 Hz, Pyr-CH), 2.45 (1H, ddd,

J = 13.5, 10.5, 5.7 Hz, PyR-CH), 3.44 (2H, d,

J = 4.6 Hz, CH₂OBn), 3.67 (1H, dt, J = 7.9, 4.6 Hz,

dioxolane 4-H), 3.75 (1H, q, J = 4.6 Hz, dioxolane 5-

H), 4.28 (1H, d, J = 12.1 Hz, CHPh), 4.54 (1H, d,

J = 12.1 Hz, CHPh), 4.64 (2H, br, NH₂), 5.14 (2H,

br, NH₂), 7.19–7.43 (10 H, m, 2· Ph-H₅); MS m/z

435.2398 (M+H) (C₂₅H₃₁N₄O₃requires 435.2396), 327

(MꢀC₇H₇O), 91 (Bn).

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4573

6.56. 6-((3S,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-

phenylpyrimidine-2,4-diamine (43a)

6.60. 1-(4-Chlorophenyl)-1-cyano-7-hydroxyheptan-2-one

(45b)/1-(4-chlorophenyl)-1-cyanohept-1-en-1,7-diol (46b)

Compound 42a was treated with aq CF₃CO₂H, as for

the synthesis of 26a, to give 43a (210 mg, 76%) as a pale

buﬀ solid: mp 101–102 ꢁC; NMR (CD₃OD) d_H1.70 (1H,

q, J = 7.6 Hz, 2⁰-H₂), 2.36 (1H, dt, J = 14.2, 7.6 Hz, 1⁰-

H), 2.48 (1H, dt, J = 14.2, 7.6 Hz, 1⁰-H), 3.42–3.55

(4H, m, 3⁰,4⁰,5⁰-H₄), 4.48 (1H, d, J = 11.7 Hz, CHPh),

4.52 (1H, d, J = 11.7 Hz, CHPh), 7.22–7.49 (10 H, m,

2· Ph-H₅); MS m/z 395.2082 (M+H) (C₂₂H₂₇N₄O₃re-

quires 359.2083), 91 (Bn).

4-Chlorophenylacetonitrile and tetrahydrooxepin-2-one

44 were treated with LiN(SiMe₃)₂, as for the synthesis

of 22a/23a, to give 45b/46b (11%) as a white solid: mp

92–94 ꢁC; NMR d_H1.21–1.30 (2H, m, 5-H₂), 1.50 (2H,

qn, J = 6.8 Hz, 6-H₂), 1.58 (2H, qn, J = 7.4 Hz, 4-H₂),

2.58 (1H, dt, J = 18.2, 7.4 Hz, 3-H), 2.66 (1H, dt,

J = 18.2, 7.4 Hz, 3-H), 3.60 (2H, t, J = 6.8 Hz, 7-H₂),

4.65 (1H, br, OH), 7.32 (2H, d, J = 8.4 Hz, Ar 2,6-H₂),

7.41 (2H, d, J = 8.4 Hz, Ar 3,5-H₂); MS m/z 268.0912

(M+H) (C₁₄H₁₇³⁷ClNO₂requires 268.0918), 266.0942

(M+H) (C₁₄H₁₇³⁵ClNO₂requires 266.0947), 250/248

(MꢀOH), 207/205 (MꢀC₃H₈O).

6.57. 6-((3S,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-(4-

chlorophenyl)pyrimidine-2,4-diamine (43b)

Compound 42b was treated with aq CF₃CO₂H, as for

the synthesis of 26a, to give 43b (75%) as a pale yellow

solid: mp 141–143 ꢁC; IR m_max3562, 3492, 3430, 3343,

1618 cm^ꢀ1; NMR (CD₃OD) d_H1.65–1.73 (2H, m, 2⁰-

H₂), 2.28 (1H, ddd, J = 13.7, 9.4, 6.6 Hz, 1⁰-H), 2.42

(1H, ddd, J = 13.7, 9.4, 6.6 Hz, 1⁰-H), 3.42-3.54 (4H,

m, 3⁰,4⁰,5⁰-H₄), 4.48 (1H, d, J = 11.7 Hz, CHPh), 4.52

(1H, d, J = 11.7 Hz, CHPh), 7.20 (2H, d, J = 8.6 Hz,

Ar 2,6-H₂), 7.30–7.36 (5H, m, Ph-H₅), 7.44 (2H, d,

J = 8.6 Hz, Ar 3,5-H₂); MS m/z 431.1680 (M+H)

(C₂₂H₂₆³⁷ClN₄O₃requires 431.1663), 429.1702 (M+H)

(C₂₂H₂₆³⁵ClN₄O₃requires 429.1693), 91 (Bn).

6.61. 1-(4-Bromophenyl)-1-cyano-7-hydroxyheptan-2-

one (45c)/1-(4-bromophenyl)-1-cyanohept-1-en-1,7-diol

(46c)

4-Bromophenylacetonitrile and tetrahydrooxepin-2-one

44 were treated with LiN(SiMe₃)₂, as for the synthesis

of 22a/23a (chromatographic eluant EtOAc/hexane,

3:1), to give 45c/46c (8%) as a pale yellow solid: mp

76–78 ꢁC; NMR d_H1.28 (2H, qn, J = 7.3 Hz, 5-H₂),

1.50 (2H, qn, J = 7.3 Hz, 6-H₂), 1.58 (2H, qn,

J = 7.3 Hz, 4-H₂), 2.62 (1H, dt, J = 18.0, 7.3 Hz, 3-H),

2.65 (1H, dt, J = 18.0, 7.3 Hz, 3-H), 3.60 (2H, t,

J = 6.4 Hz, 7-H₂), 4.64 (1H, br, OH), 7.26 (2H, d,

J = 8.2 Hz, Ar 2,6-H₂), 7.41 (2H, d, J = 8.2 Hz, Ar 3,5-

H₂); MS m/z 312.0430 (M+H) (C₁₄H₁₇⁸¹BrNO₂requires

312.0422), 310.0449 (M+H) (C₁₄H₁₇⁷⁹BrNO₂requires

310.0442), 294/292 (MꢀOH).

6.58. 6-((3S,4S)-5-Benzyloxy-3,4-dihydroxypentyl)-5-(3-

bromophenyl)pyrimidine-2,4-diamine (43c)

Compound 42c was treated with aq CF₃CO₂H, as for

the synthesis of 26a, to give 43c (95%) as a highly hygro-

scopic pale yellow solid: IR m_max3582, 3350, 1613 cm^ꢀ1

;

6.62. 1-Cyano-1-(3,4-dichlorophenyl)-7-hydroxyheptan-2-

one (45d)/1-cyano-1-(3,4-chlorophenyl)hept-1-en-1,7-diol

(46d)

NMR (CD₃OD) d_H1.71–1.78 (2H, m, 2⁰-H₂), 2.36 (1H,

dt, J = 14.6, 7.8 Hz, 1⁰-H), 2.46 (1H, dt, J = 14.6, 7.8 Hz,

1⁰-H), 3.43–3.61 (4H, m, 3⁰,4⁰,5⁰-H₄), 4.51 (1H, d,

J = 11.9 Hz, CHPh), 4.56 (1H, d, J = 11.9 Hz, CHPh),

7.18 (2H, d, J = 8.6 Hz, Ar 2,6-H₂), 7.34–7.36 (5H, m,

Ph-H₅), 7.63 (2H, d, J = 8.6 Hz, Ar 3,5-H₂); NMR

(CD₃OD) d_C29.26, 31.59, 70.80, 72.32, 71.11, 72.99,

107.30, 120.98, 122.10, 127.39, 127.58, 128.04, 130.83,

132.31, 138.05, 161.45, 161.80, 162.50; MS m/z

475.1184 (M+H) (C₂₂H₂₆⁸¹BrN₄O₃requires 475.1167),

473.1186 (M+H) (C₂₂H₂₆⁷⁹BrN₄O₄requires 473.1188),

91 (Bn).

3,4-Dichlorophenylacetonitrile and tetrahydrooxepin-2-

one 44 were treated with LiN(SiMe₃)₂, as for the synthe-

sis of 45c/46c, to give 45d/46d (25%) as a pale yellow so-

lid: mp 95–97 ꢁC; NMR d_H1.26 (2H, qn, J = 7.3 Hz, 5-

H₂), 1.53 (2H, qn, J = 7.3 Hz, 6-H₂), 1.62 (2H, qn,

J = 7.3 Hz, 4-H₂), 2.66 (2H, dt, J = 15.4, 7.3 Hz, 3-H₂),

3.60 (2H, t, J = 6.4 Hz, 7-H₂), 4.69 (1 H, br, OH), 7.39

(1H, d, J = 8.4 Hz, Ar 6-H), 7.51 (1H, d, J = 8.4 Hz,

Ar 5-H), 7.83 (1H, s, Ar 2-H); MS m/z 304.0520

(M+H) (C₁₄H₁₆³⁷Cl₂NO₂requires 304.0499), 302.0537

(M+H) (C₁₄H₁₆³⁷Cl³⁵ClNO₂requires 302.0528),

300.0559 (M+H) (C₁₄H₁₆³⁵Cl₂NO₂requires 300.0558),

286/284/282 (MꢀOH).

6.59. 1-Cyano-7-hydroxy-1-phenylheptan-2-one (45a)/

1-cyano-1-phenylhept-1-en-1,7-diol (46a)

Phenylacetonitrile and tetrahydrooxepin-2-one 44 were

treated with LiN(SiMe₃)₂, as for the synthesis of 22a/

23a, to give 45a/46a (21%) as a pale yellow solid: mp

98–99 ꢁC; IR m_max3402, 2205, 1718 cm^ꢀ1; NMR

((CD₃)₂SO) d_H1.35–1.42 (2H, m, 5-H₂), 1.44-1.51 (2H,

m, 6-H₂), 1.65 (2H, qn, J = 7.4 Hz, 4-H₂), 2.60 (2H, t,

J = 7.4 Hz, 3-H₂), 3.40 (2H, t, J = 6.2 Hz, 7-H₂), 4.36

(1H, br, OH), 7.20 (1H, t, J = 7.6 Hz, Ph 4-H), 7.30

(2H, t, J = 7.6 Hz, Ph 3,5-H₂), 7.61 (2H, d, J = 7.6 Hz,

Ph 2,6-H₂); MS m/z 232.1329 (M+H) (C₁₄H₁₈NO₂re-

quires 232.1337), 214 (MꢀOH), 185 (MꢀC₂H₆O), 115

(MꢀC₆H₁₂O₂).

6.63. (4R,5R)-4-(2-Cyano-1-oxo-2-phenylethyl)-5-hydroxy-

methyl-2,2-dimethyl-1,3-dioxolane (49a)/(4R,5R)-4-(2-

cyano-1-hydroxy-2-phenylethenyl)-5-hydroxymethyl-2,2-

dimethyl-1,3-dioxolane (50a)

Phenylacetonitrile and 2,3-O-isopropylidene-^D-erythr-

onolactone 48 were treated with LiN(SiMe₃)₂, as for

the synthesis of 22a/23a, to give 49a/50a (27%) as a pale

yellow oil: IR m_max3408, 2246, 1694 cm^ꢀ1; NMR d_H1.41

(3H, s, Me), 1.49 (3H, s, Me), 4.41 (1H, dd, J = 11.0,

3.5 Hz, CHOH), 4.48 (1H, d, J = 11.0 Hz, CHOH),

4574

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4.75 (1H, d, J = 5.5 Hz, 4-H), 4.88 (1H, m, 5-H), 7.47

(2H, t, J = 7.4 Hz, Ph 3,5-H₂), 7.61 (1H, t, J = 7.4 Hz,

Ph 4-H), 8.10 (2H, d, J = 8.6 Hz, Ph 2,6-H₂); MS m/z

276.1225 (M+H) (C₁₅H₁₈NO₄requires 276.1235), 258

(MꢀOH).

6.67. (4R,5R)-4-(2-Cyano-1-methoxy-2-phenylethenyl)-5-

hydroxymethyl-2,2-dimethyl-1,3-dioxolane (51a) and 6-

((4S,5R)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxolan-4-

yl)-5-phenylpyrimidine-2,4-diamine (52a)

Compound 49a/50a was treated with CH₂N₂, as for the

synthesis of 24a, to give 51a (85%) as a pale yellow oil:

IR m_max3492, 2209 cm^ꢀ1; NMR d_H1.44 (3H, s, 2-Me),

1.58 (3H, s, 2-Me), 3.53 (3H, s, OMe), 4.39 (1H, dd,

J = 11.0, 3.7 Hz, CHOH), 4.45 (1H, d, J = 11.0 Hz,

CHOH), 4.60 (1H, m, 5-H), 5.37 (1H, d, J = 7.4 Hz, 4-

H), 7.30–7.41 (5H, m, Ph-H₅); MS m/z 290.1388

(M+H) (C₁₆H₂₀NO₄requires 290.1392), 274 (MꢀMe),

258 (MꢀOMe). Compound 51a was treated with guani-

dine, as for the synthesis of 25a (chromatographic eluant

CH₂Cl₂/ MeOH (4:1)), to give 52a (48%) as a pale yel-

6.64. (4R,5R)-4-(2-(4-Chlorophenyl)-2-cyano-1-oxoeth-

yl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (49b)/

(4R,5R)-4-(2-(4-chlorophenyl)-2-cyano-1-hydroxyethe-

nyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (50b)

4-Chlorophenylacetonitrile and 2,3-O-isopropylidene-^D-

erythronolactone 48 were treated with LiN(SiMe₃)₂, as

for the synthesis of 22a/23a, to give 49b/50b (21%) as a

pale yellow oil: NMR d_H1.30 (3H, s, Me), 1.59 (3H,

s, Me), 4.05 (1H, dd, J = 10.1, 3.1 Hz, CHOH), 4.09

(1H, d, J = 10.1 Hz, CHOH), 4.86 (1H, m, 5-H), 4.73

(1H, d, J = 5.8 Hz, 4-H), 7.27 (2H, d, J = 8.4 Hz, Ar

2,6-H₂), 7.48 (2H, d, J = 8.4 Hz, Ar 3,5-H₂); MS m/z

312.0849 (M+H) (C₁₅H₁₇³⁷ClNO₄requires 312.0816),

310.0855 (M+H) (C₁₅H₁₆³⁵ClNO₄requires 310.0846),

294/292 (MꢀOH).

low solid: mp 214–216 ꢁC; IR m_max3492, 3465, 3422,

20

3318, 3178, 1624 cm^ꢀ1; ½aꢁ ¼ þ3:3^ꢂ(c 4, CHCl₃);

D

NMR d_H1.21 (3H, s, Me), 1.62 (3H, s, Me), 3.48 (1H,

dd, J = 12.7, 2.1 Hz, CHOH), 3.57 (1H, dd, J = 12.7,

3.3 Hz, CHOH), 3.97 (1H, m, dioxolane 5-H), 4.79

(1H, d, J = 6.6 Hz, dioxolane 4-H), 4.90 (2H, br,

NH₂), 5.16 (2H, br, NH₂), 7.10 (1H, d, J = 7.4 Hz, Ph

2-H), 7.31 (1H, d, J = 7.4 Hz, Ph 6-H), 7.41 (1H, t,

J = 7.4 Hz, Ph 4-H), 7.47 (2H, t, J = 7.4 Hz, Ph 3,5-

H₂); MS m/z 317.1622 (M+H) (C₁₆H₂₀N₄O₃requires

317.1613).

6.65. (4R,5R)-4-(2-(4-Bromophenyl)-2-cyano-1-oxoeth-

yl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (49c)/

(4R,5R)-4-(2-(4-bromophenyl)-2-cyano-1-hydroxyethe-

nyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (50c)

4-Bromophenylacetonitrile and 2,3-O-isopropylidene-^D-

erythronolactone 48 were treated with LiN(SiMe₃)₂, as

for the synthesis of 22a/23a (chromatographic eluant

EtOAc/hexane, 3:1), to give 49c/50c (38%) as a pale yel-

low oil: IR m_max3422, 2208, 1777 cm^ꢀ1; NMR d_H1.39

(3H, s, Me), 1.47 (3H, s, Me), 4.40 (1H, dd, J = 10.9,

3.7 Hz, CHOH), 4.45 (1H, d, J = 10.9 Hz, CHOH),

4.74 (1H, d, J = 5.5 Hz, 4-H), 4.87 (1H, m, 5-H), 5.57

(1H, s, CHCN), 7.27 (2H, d, J = 8.6 Hz, Ar 2,6-H₂),

7.51 (2H, d, J = 8.6 Hz, Ar 3,5-H₂); MS m/z 356.0326

(M+H) (C₁₅H₁₇⁸¹BrNO₄requires 356.0320), 354.0327

(M+H) (C₁₅H₁₇⁷⁹BrNO₄requires 354.0340), 338/336

(MꢀOH).

6.68. (4R,5R)-4-(2(4-Chlorophenyl)-2-cyano-1-meth-

oxyethenyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane

(51b) and 5-(4-chlorophenyl)-6-((4S,5R)-2,2-dimethyl-5-

hydroxymethyl-2,2-dimethyl-1,3-dioxolan-4-yl)pyrim-

idine-2,4-diamine (52b)

Compound 49b/50b was treated with CH₂N₂, as for the

synthesis of 24a, to give 51b (69%) as a pale yellow oil:

MS m/z 326.0989 (M+H) (C₁₆H₁₉³⁷ClNO₄requires

326.0973), 324.1014 (M+H) (C₁₆H₁₉³⁵ClNO₄requires

324.1002),307/305(MꢀH₂O).Compound51bwastreated

with guanidine, as for the synthesis of 52a, to give 52b

(50%) as a pale buﬀ solid: mp 172–174 ꢁC; IR m_max3497,

3459, 3433, 3396, 3217, 1613 cm^ꢀ1; NMR d_H1.24 (3 H, s,

Me), 1.63 (3H, s, Me), 1.66 (1H, br, OH), 3.47 (1H, dd,

J = 12.8, 2.3 Hz, CHOH), 3.58 (1H, dd, J = 12.8, 3.3 Hz,

CHOH), 3.98 (1H, m, dioxolane 5-H), 4.66 (2H, br,

NH₂), 4.77 (1H, d, J = 6.2 Hz, dioxolane 4-H), 4.93 (2H,

br, NH₂), 7.05 (1H, dd, J = 8.8, 2.0 Hz, Ar 2-H), 7.27

(1H, dd, J = 9.4, 2.0 Hz, Ar 6-H), 7.43 (1H, dd, J = 8.8,

2.0 Hz, Ar 3-H), 7.47 (1H, dd, J = 9.4, 2.0 Hz, Ar 5-H);

MS m/z 353.1218 (M+H) (C₁₆H₂₀³⁷ClN₄O₃requires

353.1194), 351.1236 (M+H) (C₁₆H₂₀³⁵ClN₄O₃requires

351.1223), 295/293 (MꢀC₃H₅O).

6.66. (4R,5R)-4-(2-Cyano-2-(3,4-dichlorophenyl)-1-oxo-

ethyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxolane

(49d)/(4R,5R)-4-(2-cyano-2-(3,4-dichlorophenyl)-1-

hydroxyethenyl)-5-hydroxymethyl-2,2-dimethyl-1,3-dioxo-

lane (50d)

3,4-Dichlorophenylacetonitrile and 2,3-O-isopropylid-

ene-^D-erythronolactone 48 were treated with LiN

(SiMe₃)₂, as for the synthesis of 22a/23a (chromato-

graphic eluant EtOAc/hexane, 1:1), to give 49d/50d

(22%) as a pale yellow oil: IR m_max3404, 2250, 1782

cm^ꢀ1; NMR d_H1.30 (3H, s, Me), 1.38 (3H, s, Me),

3.93 (1H, dd, J = 10.3, 3.7 Hz, CHOH), 4.00 (1H, d,

J = 10.3 Hz, CHOH), 4.70 (1H, d, J = 5.9 Hz, 4-H),

4.91 (1H, dd, J = 5.9, 3.7 Hz, 5-H), 7.33 (1H, dd,

J = 8.2, 2.0 Hz, Ar 6-H), 7.36 (1H, d, J = 8.2 Hz, Ar 5-

H), 7.58 (1H, d, J = 2.0 Hz, Ar 2-H); MS m/z 348.0411

(M+H) (C₁₅H₁₆³⁷Cl₂NO₄requires 348.0397), 346.0901

(M+H) (C₁₅H₁₆³⁷Cl³⁵ClNO₄requires 346.0906),

344.0448 (M+H) (C₁₅H₁₆³⁵Cl₂NO₄requires 344.0456),

330/328/326 (MꢀOH).

6.69. (4R,5R)-4-(2-(4-Bromophenyl)-2-cyano-1-methoxy-

ethenyl)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxolane

(51c) and 5-(4-bromophenyl)-6-((4S,5R)-2,2-dimethyl-5-

hydroxymethyl-1,3-dioxolan-4-yl)pyrimidine-2,4-diamine

(52c)

Compound 49c/50c was treated with CH₂N₂, as for the

synthesis of 24a, to give 51c (91%) as a pale yellow oil:

NMR d_H1.44 (3H, s, 2-Me), 1.48 (3H, s, 2-Me), 3.57

(3H, s, OMe), 4.40 (1H, dd, J = 10.9, 3.5 Hz, CHOH),

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

4575

4.46 (1H, d, J = 10.9 Hz, CHOH), 4.61 (1H, m, 5-H),

5.34 (1H, d, J = 7.0 Hz, 4-H), 7.36 (2H, d, J = 8.6 Hz,

Ar 2,6-H₂), 7.50 (2H, d, J = 8.6 Hz, Ar 3,5-H₂); MS m/

z 370.0481 (M+H) (C₁₆H₁₉⁸¹BrNO₄requires 370.0476),

368.0501 (M+H) (C₁₆H₁₉⁷⁹BrNO₄requires 368.0497).

Compound 51c was treated with guanidine, as for the

synthesis of 52a, to give 52c (29%) as a pale yellow solid:

mp 181–183 ꢁC; NMR ((CD₃)₂SO) d_H1.14 (3 H, s, Me),

1.48 (3H, s, Me), 3.45 (2H, d, J = 3.5 Hz, CH₂O), 4.03

(1H, dt, J = 6.4, 3.5 Hz, dioxolane 5-H), 4.73 (1H, d,

J = 6.4 Hz, dioxolane 4-H), 5.70 (2H, br, NH₂), 5.85

(2H, br, NH₂), 7.20 (1H, dd, J = 8.1, 2.0 Hz, Ar 2-H),

7.25 (1H, dd, J = 7.7, 2.0 Hz, Ar 6-H), 7.61 (1H, dd,

J = 7.7, 2.0 Hz, Ar 5-H), 7.63 (1H, dd, J = 8.1, 2.0 Hz,

Ar 3-H); NMR (CD₃)₂SO) d_C25.44, 26.37, 62.49,

76.46, 79.81, 108.35, 108.72, 121.70, 132.30, 132.35,

132.59, 133.38, 134.30, 159.21, 162.29, 163.21; MS m/z

397.0694 (M+H) (C₁₆H₂₀⁸¹BrN₄O₃requires 397.0698),

395.0712 (M+H) (C₁₆H₂₀⁷⁹BrN₄O₃requires 395.0718).

54/55 (34%) as a pale buﬀ solid: mp 53–54 ꢁC (lit.⁵⁴

mp 76–78 ꢁC) ; IR m_max2200 cm^ꢀ1; NMR ((CD₃)₂SO)

d_H2.88 (2H, t, J = 6.4 Hz, CH₂), 2.94 (2H, t,

J = 6.4 Hz, CH₂), 7.20–7.61 (10 H, m, 2· Ph-H₅),

11.70 (1H, br s, OH) MS m/z250.1240 (M+H)

(C₁₇H₁₆NO requires 250.1231), 222 (MꢀHCN), 91

(Bn).

6.72. 1-Cyano-1-phenylpropan-2-one (58)/1-cyano-1-

phenylprop-1-en-2-ol (59)

Phenylacetonitrile was condensed with ethyl acetate 58,

as for the synthesis of 22a/23a except that chromatogra-

phy was omitted and the product was recrystallised (aq

EtOH), to give 58/59 (31%) as a pale buﬀ solid: mp 87–

88 ꢁC (lit.⁵⁵mp 87–89 ꢁC); NMR d_H2.25 (3H, s, Me),

4.66 (1H, s, CHCN), 7.38–7.47 (5H, m, Ph-H₅); MS

m/z 160.0740 (M+H) (C₁₀H₁₀NO requires 160.0762),

144 (MꢀCH₃), 118 (M – C₂H₃N).

6.70. (4R,5R)-2-Cyano-4-(2-(3,4-dichlorophenyl)-1-meth-

oxyethenyl)-2,2-dimethyl-5-hydroxymethyl-1,3-dioxolane

(51d) and 5-(3,4-dichlorophenyl)-6-((4S,5R)-2,2-dimethyl-

5-hydroxymethyl-1,3-dioxolan-4-yl)pyrimidine-2,4-

diamine (52d)

6.73. 1-(4-Chlorophenyl)-1-cyanobutan-2-one (68)/1-(4-

chlorophenyl)-1-cyano-but-1-en-2-ol (64)

4-Chlorophenylacetonitrile was condensed with ethyl

propanoate 62, as for the synthesis of 22a/23a, to give

62/63 (31%) as a pale yellow solid: mp 50–51 ꢁC (lit.¹⁶

mp 50–52 ꢁC); NMR ((CD₃)₂SO) d_H1.24 (3H, t,

J = 7.4 Hz, Me), 2.62 (2H, q, J = 7.4 Hz, CH₂), 7.42

(2H, d, J = 8.8 Hz, 2,6-H₂), 7.66 (2H, d, J = 8.8 Hz,

3,5-H₂).

Compound 49d/50d was treated with CH₂N₂, as for the

synthesis of 24a, to give 51d (78%) as a pale yellow oil:

IR m_max3534, 2247, 1595 cm^ꢀ1; NMR d_H1.44 (3H, s, 2-

Me), 1.47 (3H, s, 2-Me), 3.72 (3H, s, OMe), 4.54 (1H,

dd, J = 10.7, 4.1 Hz, CHOH), 4.73 (1H, d, J = 10.7 Hz,

CHOH), 4.96 (1H, m, 5-H), 5.52 (1H, d, J = 5.9 Hz, 4-

H), 7.15 (1H, d, J = 8.5 Hz, Ar 5-H), 7.37 (1H, dd,

J = 8.5, 1.4 Hz, Ar 6-H), 7.40 (1H, d, J = 1.4 Hz, Ar 2-

H); MS m/z 360.0378 (MꢀH) (C₁₆H₁₆³⁷Cl₂NO₄requires

360.0397), 358.0433 (MꢀH) (C₁₆H₁₆³⁷Cl³⁵ClNO₄re-

quires 358.0426), 356.0452 (MꢀH) (C₁₆H₁₆³⁵Cl₂NO₄re-

quires 356.0456), 346/344/342 (MꢀMe). Compound 51d

was treated with guanidine, as for the synthesis of 52a,

to give 52d (47%) as a pale yellow solid: mp 181–

183 ꢁC; NMR (CD₃CN) d_H1.20 (3H, s, Me), 1.49

(3H, s, Me), 3.40–3.42 (2H, m, CH₂OH), 4.03 (1H, m,

5-H), 4.72 (1H, d, J = 7.0 Hz, 4-H), 5.21 (2H, br,

NH₂), 5.31 (2H, br, NH₂), 7.11 (0.5H, dd, J = 8.2, 2.0

Hz, Ph 6-H), 7.21 (0.5H, dd, J = 8.2, 2.0 Hz, Ph 6-H),

7.38 (0.5H, d, J = 2.0 Hz, Ph 2-H), 7.48 (0.5H, d,

J = 2.0 Hz, Ph 2-H), 7.60 (0.5H, d, J = 8.2 Hz, Ph 5-

H), 7.62 (0.5H, d, J = 8.2 Hz, Ph 5-H); NMR

(CD₃)₂CO) d_C24.90 (Me), 25.88 (Me), 61.93 (CH₂OH),

76.13 (CH), 79.16 (CH), 107.43 (CMe₂), 108.75 (Pyr 5-

C), 130.36 (Ph C), 131.07 (Ph CH), 131.18 (Ph C),

131.51 (Ph CH), 132.89 (Ph CH), 135.14 (Ph C),

159.37 (Pyr 2-C), 161.93 (Pyr 4-C), 162.93 (Pyr 6-C);

MS m/z 389.0778 (M+H) (C₁₆H₁₉³⁷Cl₂N₄O₃requires

389.0775), 387.0810 (M+H) (C₁₆H₁₉³⁷Cl³⁵ClN₄O₃re-

quires 387.0833), 385.0833 (M+H) (C₁₆H₁₉³⁵Cl₂N₄O₃

requires 385.0834), 331/329/327 (MꢀC₃H₅O).

6.74. Biological assay

The radial spoke assay was performed essentially as de-

scribed by Gerum et al.³⁸and Sibley et al.⁵⁶The three

yeasts were grown in media comprising 10% yeast ex-

tract, 10% peptone and 10% dextrose. Sulfanilamide

(1.0 mM, 100 lL), an inhibitor of dihydropteroate syn-

thase,⁵⁷was spread onto fresh agar plates and allowed

to absorb into the medium overnight. Three template

plates were streaked with the yeast cultures in two

orthogonal lines and incubated at 30 ꢁC for 3 d. These

plates were used to generate replica test plates. Test

compounds 7a–d, 8a–c, 9a–d, 10a–d and control com-

pounds 3, 6, 11, 12 were made up as 10 mM solutions

in DMSO; a spot (10 lL) of each of these solutions

was placed at the centre of each test plate. The assay

plates were then incubated for 3 days at 30 ꢁC before

the inhibition zone was measured. Each compound/

yeast combination was assayed in triplicate.

Acknowledgments

We thank Dr. Steven J. Black and Dr. Timothy Wood-

man (University of Bath) for the NMR spectra, Jo Car-

ter (University of Bath) for support of the

microbiological study, the EPSRC Mass Spectrometry

Centre (Swansea) for some of the mass spectra and

Dr. Carol Hopkins Sibley (University of Washington)

for the kind gift of the yeast cell lines. We are very grate-

ful to the Government of the Arab Republic of Egypt

for a studentship (to MHRIE-H).

6.71. 1-Cyano-1,4-diphenylbutan-2-one (54)/1-cyano-1,4-

diphenylbut-1-en-2-ol (55)

Phenylacetonitrile was condensed with ethyl 3-phenyl-

propanoate 53, as for the synthesis of 22a/23a, to give

4576

M. H. R. I. El-Hamamsy et al. / Bioorg. Med. Chem. 15 (2007) 4552–4576

30. da Cunha, E. F. F.; Ramalho, T. C.; de Alencastro, R.

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