Analogues of 4-[(7-Bromo-2-methyl-4-oxo-3 H -quinazolin-6-yl)methylprop-2- ynylamino]- N -(3-pyridylmethyl)benzamide (CB-30865) as potent inhibitors of nicotinamide phosphoribosyltransferase (Nampt)

Article abstract of DOI:10.1021/jm101145b

We have shown previously that the target of the potent cytotoxic agent 4-[(7-bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methyl-prop-2-ynylamino] -N-(3-pyridylmethyl)benzamide (CB38065, 1) is nicotinamide phosphoribosyltransferase (Nampt). With its cellular target known we sought to optimize the biochemical and cellular Nampt activity of 1 as well as its cytotoxicity. It was found that a 3-pyridylmethylamide substituent in the A region was critical to cellular Nampt activity and cytotoxicity, although other aromatic substitution did yield compounds with submicromolar enzymatic inhibition. Small unsaturated groups worked best in the D-region of the molecule, with 3,3-dimethylallyl providing optimal potency. The E region required a quinazolin-4-one or 1,2,3-benzotriazin-4-one group for activity, and many substituents were tolerated at C² of the quinazolin-4-one. The best compounds showed subnanomolar inhibition of Nampt and low nanomolar cytotoxicity in cellular assays.

Full text of DOI:10.1021/jm101145b

8734 J. Med. Chem. 2010, 53, 8734–8746
DOI: 10.1021/jm101145b
Analogues of 4-[(7-Bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methylprop-2-ynylamino]-
N-(3-pyridylmethyl)benzamide (CB-30865) as Potent Inhibitors of Nicotinamide
Phosphoribosyltransferase (Nampt)
Jeffrey W. Lockman,*,† Brett R. Murphy,† Daniel F. Zigar,† Weston R. Judd,† Paul M. Slattum,† Zhong-Hua Gao,‡
Kirill Ostanin,‡ Jeremy Green,‡ Rena McKinnon,‡ Ryan T. Terry-Lorenzo,‡ Tracey C. Fleischer,‡ J. Jay Boniface,‡
Mark Shenderovich,† and J. Adam Willardsen†
†Department of Medicinal Chemistry, and ‡Department of Discovery Biology, Myrexis, Inc., 305 Chipeta Way, Salt Lake City,
Utah 84108, United States
Received September 3, 2010
We have shown previously that the target of the potent cytotoxic agent 4-[(7-bromo-2-methyl-4-oxo-
3H-quinazolin-6-yl)methyl-prop-2-ynylamino]-N-(3-pyridylmethyl)benzamide (CB38065, 1) is nicotin-
amide phosphoribosyltransferase (Nampt). With its cellular target known we sought to optimize the
biochemical and cellular Nampt activity of 1 as well as its cytotoxicity. It was found that a 3-pyridyl-
methylamide substituent in the A region was critical to cellular Nampt activity and cytotoxicity,
although other aromatic substitution did yield compounds with submicromolar enzymatic inhibi-
tion. Small unsaturated groups worked best in the D-region of the molecule, with 3,3-dimethylallyl
providing optimal potency. The E region required a quinazolin-4-one or 1,2,3-benzotriazin-4-one
group for activity, and many substituents were tolerated at C2 of the quinazolin-4-one. The best
compounds showed subnanomolar inhibition of Nampt and low nanomolar cytotoxicity in cellular
assays.
Introduction
We have previously reported the direct target affinity puri-
fication (DTAP) of solid-phase linked analogues of 2.4 These
chemical proteomics-based studies revealed that the target of
1 and 2 is nicotinamide phosphoribosyltransferase (Nampt,
NAMPRTase, EC 2.4.2.12). Compound 2 inhibits Nampt in
an enzymatic assay with an average IC50 value of 11 nM.4
Mechanism-based cellular assays confirmed the cellular inhibi-
tion of Nampt by 2: compound 2 causes cellular nicotinamide
adenine dinucleotide (NAD) levels to drop, as measured directly
and by concomitant loss of poly(ADP-ribose) polymerase
(PARP) activity. The drop in cellular NAD levels, PARP
inhibition, and the cytotoxicity caused by 2 are completely
rescued by the addition of exogenous nicotinic acid, further
supporting Nampt inhibition as the mechanism of action of 2.
Nampt catalyzes the first and rate-limiting step in the
conversion of nicotinamide (NAM) to NAD (Figure 2). This
salvage pathway is the only way a mammalian cell can recycle
the NAM formed during NAD consumption by enzymes such
as PARPs and sirtuins.5 Nampt is critical to cell viability, as
NAD is an essential cofactor in ATP generating processes.
Cancer cells have both increased demand for ATP and
increased activity of NAD-consuming enzymes, and conse-
quently Nampt has emerged as an attractive target for the
development of anticancer therapeutics.6 Phase I clinical re-
sults have been reported for two Nampt inhibitors (FK866, 3,
and GMX1778/CHS-828, 4),7b and phase II studies on each
are ongoing. More recently 1,2,3-triazole-containing com-
pounds have been reported to be potent Nampt inhibitors:
compound 5 depletes cellular NAD levels with IC50=3.0 nM
and shows an SH-SY5Y cytotoxicity of 3.8 nM.8
The quinazolinone cytotoxic agent CB30865 (1, Figure 1a)
was developed as part of an effort to improve folate-based
inhibitors of thymidylate synthase (TSa) by replacing the
classical antifolate terminal glutamic acid residue with ami-
nomethylpyridine moieties.1 The stated goal of this replace-
ment was to increase the lipophilicity and consequently the
cell permeability of these compounds and to prevent unde-
sirable intracellular polyglutamation. The 2-, 3-, and 4-pyr-
idylmethylamides were moderately potent inhibitors of
mammalian TS (IC50 = 156-508 nM), but the 3-amino-
methylpyridyl analogue (1) showed dramatically increased
W1L2 cytotoxicity (EC50=2.8 nM) that was not rescued by
precursors or end-products of folate metabolism.2 These
data and the cytotoxicty profile of 1 across a wide range of
cell lines suggested a folate-independent mechanism of ac-
tion. Water-soluble analogues of 1 were made in an effort
to improve in vivo efficacy, and the best of these com-
pounds showed subnanomolar W1L2 cytotoxicity (2, EC50 =
0.49 nM).3
*To whom correspondence should be addressed. Phone: 801-214-
7885. Fax: 801-214-7993. E-mail: jeffrey.lockman@myrexis.com.
a Abbreviations: TS, thymidylate synthase; DTAP, direct target affinity
purification; Nampt, nicotinamide phosphoribosyltransferase; NAD, nico-
tinamide adenine dinucleotide; PARP, poly(ADP-ribose) polymerase;
NAM, nicotinamide; ATP, adenosine triphosphate; NMN, nicotinamde
mononucleotide; Naprt, nicotinic acid phosphoribosyltransferase; DHFR,
dihydrofolate reductase; NBS, N-bromosuccinimide; AIBN, 2,20-azobis(2-
methylpropionitrile); DIC, N,N0-diisopropylcarbodiimide; HOBt, 1-hydro-
xybenzotriazole; DPPA, diphenylphosphorylazide; TEPP, tetraethyl pyro-
phosphate.
r
pubs.acs.org/jmc
Published on Web 11/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8735
Figure 1. (a) Previously reported Nampt inhibitors. (b) Division of 1 for study.
Figure 2. Role of Nampt in the maintanance of cellular NAD levels. Nampt acts as the rate-limiting step in the recycling of NAM formed by
NAD consumption by PARPs and sirtuins. When Nampt is inactivated, NAD can be generated in cells expressing Naprt via a compensatory
pathway starting with exogenous nicotinic acid. The figure is adapted from ref 5c.
With the enzymatic target of 1 and its water-soluble ana-
logue 2identified, we undertook efforts to optimize their activity
toward Nampt inhibition and cytotoxicity. We divided com-
pound 2 into five structural regions (Figure 1b). The cocrystal
structure of 3 in Nampt defined the binding site as roughly
cylindrical, with the nicotinamide-mimicking pyridyl moeity
buried inside and the distal E-region solvent exposed.9 The
narrow shape of this cavity precluded much SAR in the
central portion of the inhibitor, and consequently we focused
our efforts on the A, D, and E regions. Thymidylate synthase
inhibition is known to be abrogated by alkylation at N3 of the
quinazolinone,3 and we consequently sought to retain the
N3-methyl group of 2 as much as possible.
Chemistry
The previously reported synthesis of compound 23 was
optimized to allow for the large scale (>100 g) synthesis of key
intermediate 5-methyl-6-chloroanthranilic acid (10b, Scheme 1).
The protection of 4-methyl-5-chloroaniline with acetic anhy-
dride in EtOAc/pyridine was followed by electrophilic bromine/
acetic acid bromination. Lithium-halogen exchange followed
by carbon dioxide quench and deprotection led to anthranilic

8736 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Lockman et al.
Scheme 1a
a Reagents: (a) Ac2O, pyr, EtOAc; (b) Br2, AcOH; (c) (i) n-BuLi, hexanes/THF, (ii) CO2 (g); (d) NaOH, H2O.
Scheme 2a
a Reagents: (a) Ac2O; (b) MeNH2-HCl, DMF; (c) NBS, AIBN or (PhCOO)2, CCl4, hν; (d) HCONHMe; (e) MeNH2-HCl, DIC, HOBt, DIEA,
DCM; (f) NaNO2, HCl, H2O; (g) Na, EtOH, ClCH2CN; (h) KOAc, DMF; (i) NaH, MeI, DMF.
acid 10b. This carboxylation protocol was found to be much
higher yielding than the cyanation-hydrolysis procedure
described previously.3
4-one, and chloride displacement with potassium acetate
followed by NaH/iodomethane methylation yielded ester 17.
We found that esterification with potassium acetate pro-
ceeded in much higher yield than described previously3 with
cesium acetate. The quinazolin-4-one ester 17 was brominated
analogously to 11a/b to yield bromide 18.
Quinazolin-4-one and 1,2,3-benzotriazin-4-one syntheses
(Scheme 2) each began with commercially available 5-methyl-
anthranilic acid (10a) or 4-chloro-5-methylanthranilic acid
10b. For 2-methylquinazolin-4-ones 11a and 11b, anthranilic
acid 10a or 10b was heated at reflux in acetic anhydride fol-
lowed by treatment of the resulting 2-methyl-3,1-benzoxazin-
4-one with N-methylamine hydrochloride in DMF. Anthranil-
ic acid 10a or 10b was heated neat in N-methylform-
amide10 to yield quinazolinones 13a and 13b. Anthranilic acid
10b was allowed to react with N-methylamine hydrochloride
in the presence of DIC/HOBt/DIEA/DCM and the resulting
anthranilamide treated with sodium nitrite to give 1,2,3-ben-
zotriazin-4-one 15.11 Bromination with NBS using catalytic
AIBN or dibenzoyl peroxide in carbon tetrachloride led to
bromomethyl compounds 12a/b, 14a/b, and 16. Irradiation of
the reaction mixture with UV light was found to be critical to
this bromination. The synthesis of methylene-linked E regions
proceeded largely as described in the literature.3 Anthranilic
acid 10b was treated with sodium metal and chloroacetonitrile
to generate 7-chloro-2-(chloromethyl)-6-methyl-3H-quinazolin-
Esters of 4-aminobenzoic acid were alkylated with the appro-
priate D-region bromide in the presence of 2,6-lutidine in DMF
followed by reaction with 2-chlorobenzyl bromide or bromo-
methyl heterocycle 12, 14, 16, or 18 (Scheme 3). Amides were
made using the appropriate acid chloride and Et3N in DCM
followed by alkylation as described above. Esters 19a-k and
21a-e were deprotected and coupled with 3-aminomethylpyr-
idine in the presence of DIC/HOBt/DIEA in DCM to yield
20a-k, 22a-p, and 23a-s. It was found that if amide coupling
preceded the addition of the bromomethyl heterocycle, alkyl-
ation occurred on the pyridyl nitrogen of 24 to yield quaternary
pyridinium salt 25 rather than the desired N-alkylated aniline.
These salts were found to be inactive against Nampt.
Functionalization at the C2 methylene of ester 23r also
necessitated changes to literature procedures (Scheme 3).
Whereas hydrolysis of acetate 23r was previously described
using aqueous sodium hydroxide, in our hands these conditions

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8737
Scheme 3a
a See Tables 1-3 for A, D, and E substituents. Reagents: (a) D-Br, 2,6-lutidine, DMF or RCOCl, Et3N, DCM; (b) E-Br (2-chlorobenzyl bromide, 12,
14, 16, or 18), 2,6-lutidine, DMF; (c) NaOH, EtOH/H2O or TFA/DCM; (d) A-NH2, DIC, HOBt, DIEA, DCM; (e) H2SO4, MeOH; (f) Ms2O, Et3N,
DCM; (g) HNR1R2.
Scheme 4a
a Reagents: (a) KMnO4, pyr/H2O; (b) 4-amino-N-(3-pyridylmethyl)benzamide, TEPP, PhCH3; (c) DPPA, Et3N, t-BuOH; (d) TFA, DCM; (e)
4-carboxybenzoic acid, NaBH(OAc)3, TFA, i-PrOAc; (f) 3-aminomethylpyridine, DIC, HOBt, DIEA, DCM.
resulted in the opening of the quinazolinone ring, yielding an
acylated anthranilic acid derivative. Acidic hydrolysis of the
acetate was successfully performed with H2SO4/MeOH. Al-
cohol 26 was converted into the methanesulfonate ester with
Ms2O and displaced with amines to yield C2 aminomethyl
compounds 2 and 27.
(13) to carboxylic acid 28 with potassium permanganate
(Scheme 4). Standard carbodiimide coupling of acid 28 to
the 3-aminomethylpyridylamide of 4-aminobenzoic acid was
unsuccessful, and consequently TEPP was utilized to produce
amide 29. Coupling with secondary anilines such as 24 were
also unsuccessful regardless of coupling agent employed. To
generate the reverse amine linkage in the D-region, carboxylic
acid 28 was allowed to react with diphenylphosphorylazide
Additional changes to the D-region of the molecule were
achieved by oxidation of 7-chloro-3,6-dimethylquinazolin-4-one

8738 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Lockman et al.
Scheme 5a
a Conditions: (a) chloroacetone, DMF; (b) I2, 4-mercaptobenzoic acid, EtOH/H2O; (c) RNH2, DIC, HOBt, DIEA, DCM; (d) hydroxyacetone, Et3N,
MeOH; (e) guanidine-HCl, NaOEt/EtOH; (f) tert-butyl bromoacetate, 2,6-lutidine, DMF; (g) TFA/DCM; (h) (COCl)2, DCM/DMF; (i) (i)
TMSCHN2, Et2O, (ii) HCl (g); (j) 2,6-diamino-4-pyrimidone, DMF; (k) paraformaldehyde, 3-ethylthiazoline bromide, Et3N, EtOH; (l) malononitrile,
Et3N, MeOH; (m) guanidine-HCl, KOEt, EtOH; (n) 3-aminomethylpyridine, DIC, HOBt, DIEA, DMF; (o) guanidine-HCl, Na, EtOH; (p) ethyl
4-isocyanatobenzoate, DCM; (q) LiOH, H2O; (r) 3-aminomethylpyridine, EDCI, HOBt, Et3N, DMF; (s) 4-(3-pyridylmethylcarbamoyl)benzoic acid,
EDCI, HOBt, Et3N, DMF.
(DPPA) in tert-butanol under Curtius conditions12 and the
resulting Boc-protected amine deprotected with TFA. Reduc-
tive amination13 of 5-aminoquinazolinone 30 with 4-formyl-
benzoic acid followed by DIC/HOBt coupling with 3-amino-
methylpyridine led to reverse amine 31.
More dramatic changes to the quinazolinone core were
made using literature methods reported for the synthesis of
thymidylate synthase and dihydrofolate reductase inhibitors
(Scheme 5). The condensation of 2,6-diamino-4-pyrimidone
with chloroacetone generated a mixture of 2-amino-4-oxo-6-
methylpyrrolo[2,3,d]pyrimidine (32) and 5-methylfuro[2,3-
d]pyrimidine-2,4-diamine (33).14b The reaction of heterocycle
32 with iodine and 4-mercaptobenzoic acid followed by DIC/
HOBt/DIEA-mediated amidation with 3-aminomethylpyri-
dine yielded compound 35.14 Malononitrile was condensed
with hydroxyacetone followed by guanidine hydrochloride to
generate 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine (36),
which was then allowed to react with iodine and 4-mercapto-
benzoic acid followed by DIC/HOBt/DIEA-mediated amida-
tion to give compounds 37 and 38.15
Aniline 24 was alkylated with tert-butyl bromoacetate,
deprotected with TFA/DCM, and the resulting acid was con-
verted into the corresponding acid chloride via oxalyl chloride
activation. A diazoketone was generated in situ through re-
action with TMS-CHN2 and quenched with HCl gas to yield
R-chloroketone 39.17 R-Chloroketone 39 underwent ring
closure with 2,6-diamino-4-pyrimidone to yield furo[2,3-d]-
pyrimidine 40.14 The expected side product of 2-amino-4-oxo-
5-alkylated-pyrrolo[2,3,d]pyrimidine was not isolated. Ethyl
4-(3-oxopropyl)benzoate was treated with paraformaldehyde
in the presence of Et3N and 3-ethylbenzothiazoline bromide
and the resulting R-hydroxy ketone condensed with malono-
nitrile. Ring closure with guanidine hydrochloride and con-
comitant ester hydrolysis produced acid 41. This acid was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8739
Table 1. A-Region Substitutiona
Figure 3. Compound 2 docked into Nampt active site. The D-region
propargyl group is positioned in a cleft bordered by Tyr188, His191,
and Tyr240.
nicotinicacid was measuredin selected compounds toconfirm
Nampt inhibition as the basis of cell killing.
The cocrystal structure of an analogue of 3 with Nampt9b
shows its pyridyl ring in the substrate binding pocket where it
is capable of interacting with the protein in two possible ways:
a salt bridge can be formed between the pyridyl nitrogen and
the acidic hydrogens of Asp219 or Asp16, and the pyridyl ring
ispositionedbetweenPhe193 andTyr18whichcould allowfor
a π-stacking interaction. To gauge the importance of these
interactions, the A-region was substituted with a variety of
aromatic, heteroaromatic, and aliphatic substituents.
The A-region was found to be rather intolerant of substitu-
tion, as only the 3-pyridyl moiety of 1 was found to give sub-
nanomolar Nampt inhibition or low nanomolar 3-day HCT116
cytotoxicity (Table 1). Moderate activity was observed with a
1H-benzimidazol-2-ylmethylamide, but other basic function-
alities such as aliphatic and anilinic amines (exemplified by
22g) showed complete inactivity at 10 μM. Inhibition was seen
with a benzimidazole A-region regardless of the rest of the
molecule. The activity of benzimidazole derivatives 22a, 22d,
22f, and 22p was comparable to that of the 2-pyridyl analogue
of 2 (IC50=170 nM),4 suggesting that while a 3-pyridyl sub-
stituent is optimally situated to participate in a salt bridge with
Asp16 or Asp219 a basic heteroaromatic group positioned
differently can also participate but to a lesser degree. Weak
biochemical activity was also seen with several A-region aro-
matic groups. Electron-deficient rings were generally more
potent than electron-rich systems, supporting the involvement
of π-stacking interactions, although 3,4-methylenedioxy sub-
stitution (22h) also showed good inhibition. The importance
of π-stacking is also suggested by the complete inactivity of
3-piperidyl analogue 22g.
a R = P: propargyl. R= D: 3,3-dimethylallyl. n/d = not done.
coupled with 3-aminomethylpyridine in the presence of DIC/
HOBt/DIEA to generate compound 42.16
Lastly, 5,6,7,8-tetrahydro-3H-pyrido[4,3-d]pyrimidin-4-one
core 43 was made through the condensation of ethyl 4-oxopi-
peridine-3-carboxylate with guanidine hydrochloride in the
presence of NaOEt/EtOH. Urea formation with heterocycle
43 and ethyl 4-isocyanatobenzoate followed by ester hydrolysis
with lithium hydroxide and peptide coupling with 3-amino-
methylpyridine yielded 44. Amidation of 43 with 4-(3-pyridyl-
methylcarbamoyl)benzoic acid produced compound 45.
It is noteworthy that while the 2-pyridyl analogue of 1showed
cytotoxicty commensurate with its biochemical inhibition,4 all
other analogues in this series with similar inhibitory activities
were almost completely inactive in cells. Preliminary data
show that while these compounds inhibited Nampt with sub-
micromolar potencies, the maximum inhibition of Nampt was
approximately 90% compared to that of untreated enzyme
(data not shown). Compounds that exhibit cytotoxicity and
cellular Nampt inhibition typically showed biochemical Nampt
inhibition of 95-96%. Additional studies are underway to
Biological Evaluation and Discussion
Compounds were screened in a coupled-enzyme assay that
monitored the Nampt-catalyzed conversion of NAM to
NMN.4 Cytotoxicity was determined by monitoring ATP
levels after 72 h of treatment of HCT116 colon carcinoma
cells. Cellular inhibition of Nampt was measured by the loss of
PARP activity after H2O2-induced DNA damage in MCF-
10A cells stably transduced with the PIK3CA(H1047R)
oncogene.4 Cytotoxicity in the presence of 10 μM exogenous

8740 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Table 2. D-Region Substitutiona
Lockman et al.
quinazolinone series. Straight-chain aliphatic groups increased
in activity according to their size up to C3 (Me, Et < Pr . Bu),
and branching was tolerated at C3 but not C2. Substitution by
unsaturated groups containing three to four carbons was found
to be ideal, and a 3,3-dimethylallyl moiety was the most potent
compound tested in this series (20i, HCT116 EC50 =16 nM).
Benzyl substitution at this position (20k) was shown to be too
large, giving a 10-fold reduction in activity compared to the
most active substituents in the biochemical, cellular, and cyto-
toxicity assays.
The best of these groups was then synthesized within
the quinazolinone series. As with the simplified system, 3,3-
dimethylallyl (22c, 22o, 23b, 23c) showed the strongest in-
hibitory activities across all quinazolinone series with the best
compounds showing cytotoxicity EC50 values less than 10 nM.
In addition propargyl substitution (22b, 22e, 23a) yielded
compounds with activity similar to that of 3,3-dimethylallyl.
Modeling suggested that the polarizable electrons of the
unsaturated system could interact in an edge-to-face fashion
with all three aromatic residues bordering the binding pocket
(Tyr188, His191, and Tyr240), leading to increased activity
over aliphatic groups of similar length (Figure 3). An alterna-
tive explanation for the activity of propargyl and 3,3-dimethyl-
allyl analogues is that the cleft is simply too narrow to ac-
commodate a sp3-hybridized carbon past the β position
relative to the nitrogen while a more flat sp2 or sp system fills
the cavity without steric clashes.
To distinguish between these possibilities, additional ana-
logues were synthesized. The most success was found with
methylcyclopropyl (23d, 23k) and homopropargyl (23f, 23j)
substitution, while methylacetamide (23e) and 3,5-dimethyl-
4-isoxazole(23h) substitution gave compounds with moderate
activity. Compound 23h showed no cytotoxicity despite sub-
100 nM potency against the enzyme. The biochemical activity
of 23h despite its considerable size downplays the potential
involvement of sterics, although its lack of cytotoxicity sug-
gests that sterics play some role. Nitrogen acylation led to
inactive compounds, where modeling predicted an unfavor-
able steric clash at the D-region side chain R-position. Lastly,
more radical changes were undertaken in the D-region.
Reversing the benzylamine group (31) resulted in moderate
inhibitory activity, as did replacement of the benzylamine
linkage with a benzamide (29). These compounds did show
cytotoxicity commensurate with their biochemical activities.
Variation around the E-region of 1 led to extremely potent
compounds when a heteroaromatic system with two six-
membered rings was retained (Table 3). Addition of the 3-amino-
propyl linker to 2 led to a 40-fold reduction in cytotoxicity
(27); however, much of the activity was regained by replace-
ment of the N-methylpiperazine moiety with either a hydroxyl
group (26) or a proton (22b). The lower than expected cyto-
toxicity of 27 compared to its biochemical potency was likely
the result of its high polarity and concomitant low cellular
permeability. Much less variation in activity was demonstrated
in the 3,3-dimethylallyl series, as low nanomolar cytotoxicities
were observed with 2-unsubstituted and 2-methyl analogues
(22c, 23c), as well as with the 1,2,3-triazolinone (22q).
a n/d = not done.
investigate the connection between this higher maximum
inhibitory effect and cellular activity.
Molecular modeling of 2 suggested that the D-region
substituent binds into a cleft defined by Tyr188, His191, and
Tyr240 of the Nampt peptide(Figure3). Toexplorethe nature
of this cleft, compounds with varying D-regions were synthe-
sized and screened (Table 2). For initial D-region exploration
a synthetically simplified series was constructed wherein the
quinazolinone portion of 2 was replaced by a 2-chlorophenyl
group. These compounds generally showed significantly re-
duced Nampt inhibition and cytotoxicity compared to the
As more dramatic changes to the quinazolinone core are
known to be tolerated in both inhibition of TS and DHFR,13-15
several other heterocycles were synthesized and screened
against Nampt. While a number of these compounds showed
submicromolar activity against Nampt in the biochemical
assay, none showed significant cellular Nampt activity or
cytotoxicty at 10 μM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8741
Table 3. E-Region Substitutiona
activity in the E-region of the molecule, although a variety of
substituents at the C2 position are tolerated. Several Nampt
inhibitors with low nanomolar biochemical, cellular, and
cytotoxicity potencies were synthesized. Studies are underway
to further characterize their cellular and in vivo effects.
Experimental Section
General Methods and Materials. The 1H NMR spectra were
recorded at 400 MHz. Chemical shifts are reported in parts per
million (ppm) downfield from TMS (0.00 ppm), and J coupling
constants are reported in hertz. HPLC-MS experiments were
run in the ESI mode using an Xterra MS C18 (Waters) 4.6 mm ꢀ
50 mm, 5 μm column. HPLC purity was determined using a
4.6 mm ꢀ150 mm Xterra C18 5 μm column, observing at both
203 and 280 nm. Both HPLC-MS and HPLC were reverse
phase with an AcCN/H2O (0.01% v/v TFA) gradient and a flow
rate of 0.5 mL/min. All final compounds were g95% pure by
HPLC at both 203 and 280 nm. MPLC purifications were
performed using an Isco RF system utilizing either a hexane/
EtOAc or DCM/MeOH gradient. The synthesis of compounds
2 and 27 has been previously described.4
N-(2-Bromo-5-chloro-4-methylphenyl)acetamide3 (8). Acetic
anhydride (142 mL, 1.5 mol, 1.5 equiv) is added slowly to
3-chloro-4-methylaniline (142 g, 1 mol) and pyridine (120 mL,
1.5 mol, 1.5 equiv) in 1 L of ethyl acetate at 0 °C. The solution
was allowed to warm to room temperature over 90 min and
washed with 4 ꢀ 200 mL of 10% aqueous HCl. The organic layer
was dried with Na2SO4 and concentrated. Acetic acid (1 L) was
added and the solution fitted with a mechanical stirrer. Bromine
(96 mL, 1.9 mol, 1.9 equiv) in 100 mL of acetic acid was added
dropwise at 0 °C and the solution allowed to warm to room tem-
perature overnight. The suspension was filtered and the filtrate
concentrated. DCM (1 L) was added to the resulting solid, and
NaHCO3 was added until the solid dissolved. The solution was
washed with water, 10% Na2S2O3, dried with Na2SO4, and
concentrated to approximately 300 mL. The resulting suspen-
sion was filtered and the filtrate washed with saturated NaH-
CO3, dried with Na2SO4, and concentrated to approximately
100 mL. The resulting suspension was filtered again and solid
combined with the first fraction of solid. The combined solids
were dried under vacuum to yield 221 g of white solid (84%).
The solid was carried on without further purification.
2-Amino-4-chloro-5-methylbenzoic Acid3 (10b). n-BuLi (2.5 M
in hexanes, 440 mmol, 2.2 equiv) is added dropwise at -78 °C
over 2.5 h to bromide 8 (52.5 g, 200 mmol) in 1 L of THF. The
solution is stirred at -78 °C for 15 min, and CO2 is bubbled
through the solution as it warmed from -78 °C to room
temperature overnight. A small amount of MeOH is added
and the solution concentrated. DCM (600 mL) is added and the
solution washed with 10% aqueous HCl, dried with Na2SO4,
and concentrated. The resulting solid is triturated with DCM to
yield 24.32 g of white solid (9, 54%). A 5 M solution of sodium
hydroxide (110 mL, 5 equiv) is added and the reaction heated at
reflux overnight. The solution is cooled, and concentrated HCl
is added to obtain pH 4-5. The resulting solid is filtered and
dried under vacuum and 17.5 g of white solid isolated (87%).
2,3,6-Trimethylquinazolin-4-one (11a). Acetic anhydride
(0.5 mL/mmol) is added to 5-methylanthranilic acid and the
suspension heated at reflux for 1 h, cooled to room temperature,
and filtered. The solid is triturated with Et2O. Methylamine
hydrochloride (3 equiv) and DMF (1 M) are added, and the
solution is heated at 120 °C overnight. Ethyl acetate is added,
washed with saturated NaHCO3, dried with Na2SO4, and con-
centrated. 11b is synthesized analogously from 8.
a R = P: propargyl. R= D: 3,3-dimethylallyl. n/d = not done.
In all compounds tested the mechanism-based cellular
PARP assay results were consistent with both Nampt inhibi-
tion and HCT116 cytotoxicity. Compounds with A-region
variation that possess biochemical potency but lack HCT116
cytotoxicity show a similar inactivity in the PARP assay.
Lastly, the cytoxicity of several potent analogues (20f, 20g,
20i, 22c, 23c, and 23e) was rescued (EC50 > 10 μM) when
tested in the presence of 10 μM exogenous nicotinic acid.
These data strongly suggest that Nampt inhibition was the
primary cause of their cytotoxicity.
In conclusion, a series of Nampt inhibitors was designed
and synthesizedbasedonthequinazolin-4-one1 and itswater-
soluble analogue 2. A 3-substituted pyridine group in the
A-region was required for significant HCT116 cytotoxicity,
although weakly potent Nampt inhibitionwas achieved witha
variety of aromatic substitution. A small unsaturated group in
the D-region was optimal for both Nampt inhibition and
cytotoxicity. Polarizable electrons that can interact in an edge-
to-face manner with Tyr188, His191, or Tyr240 of the Nampt
protein are implicated by modeling to be the cause of this
preference, although simple sterics cannot be discounted. A
quinazolin-4-one or 1,2,3-benzotriazin-4-one is required for
6-(Bromomethyl)-2,3-dimethylquinazolin-4-one (12a). Quina-
zoline 11, NBS (1.1 equiv), and AIBN (5 mol %) in CCl4 (0.2 M)
are heated at reflux under a UV lamp until reaction is complete
by LC/MS. The solution is washed with 10% aqueous sodium
bisulfite, dried with Na2SO4, and concentrated. The resulting

8742 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Lockman et al.
solid is purified by MPLC (0-100% EtOAc/hexanes). 12b is
made analogously from 11b.
3.01 (t, J=7.0 Hz, 2H), 1.55-1.46 (m, 2H), 1.41-1.30 (m, 2H),
0.90 (t, J = 7.4 Hz, 3H). HRMS [M þ H] calculated for
C24H26ClN3O 408.183 72, found 408.187 43.
3,6-Dimethylquinazolin-4-one (13a). 5-Methylanthranilic acid
is heated in N-methylformamide (4 equiv) at 180 °C for 2 h. The
solution is cooled, diluted with EtOAc, washed with 1 M NaOH,
dried with Na2SO4, and concentrated. 13b is synthesized analo-
gously from 8.
4-[(2-Chlorobenzyl)(2-methylpropyl)amino]-N-(pyridin-3-yl-
methyl)benzamide (20e). 1H NMR (DMSO-d6) δ: 8.77 (t, J=6.0 Hz,
1H), 8.68 (br s, 1H), 8.63 (d, J=4.8 Hz, 1H), 8.07 (d, J=8.0 Hz, 1H),
7.70-7.67 (m, 3H), 7.49 (dd, J=1.6, 8.0 Hz, 1H), 7.30-7.22 (m,
2H), 6.96 (dd, J=1.6, 7.6 Hz, 1H), 6.63 (d, J=9.2 Hz, 2H), 4.69 (s,
2H), 4.51 (d, J=6.0 Hz, 2H), 3.35 (d, J=7.6 Hz, 2H), 2.13-2.06 (m,
1H), 0.95 (s, 3H), 0.93 (s, 3H). HRMS [M þ H] calculated for
C24H26ClN3O 408.183 72, found 408.183 42.
4-[(2-Chlorobenzyl)(3-methylbutyl)amino]-N-(pyridin-3-yl-
methyl)benzamide (20f). 1H NMR (DMSO-d6) δ: 8.78 (t, J=6.0
Hz, 1H), 8.66 (d, J=2.0 Hz, 1H), 8.60 (dd, J=1.6, 5.6 Hz, 1H),
8.04 (dt, J=8.4, 2.0 Hz, 1H), 7.70 (d, J=9.2 Hz, 2H), 7.65 (dd,
J=5.2, 8.0 Hz, 1H), 7.50 (dd, J=1.6, 8.0 Hz, 1H), 7.32-7.24 (m,
2H), 7.03 (dd, J=2.0, 7.6 Hz, 1H), 6.59 (d, J=8.8 Hz, 2H), 4.63
(s, 2H), 4.51 (d, J = 6.0 Hz, 2H), 3.48 (t, J = 7.6 Hz, 2H),
1.67-1.58 (m, 1H), 1.50 (q, J=6.8 Hz, 2H), 0.93 (s, 3H), 0.91 (s,
3H). HRMS [M þ H] calculated for C25H28ClN3O 422.199 37,
found 422.200 43.
4-[(2-Chlorobenzyl)(prop-2-yn-1-yl)amino]-N-(pyridin-3-yl-
methyl)benzamide (20g). 1H NMR (DMSO-d6) δ: 8.86 (t, J=5.9
Hz, 1H), 8.63 (d, J=15.0 Hz, 2H), 8.00 (d, J=6.8 Hz, 1H), 7.76
(d, J=9.0 Hz, 2H), 7.61 (dd, J=7.8, 5.1 Hz, 1H), 7.51 (d, J=7.4
Hz, 1H), 7.34-7.24 (m, 2H), 7.18 (d, J=7.0 Hz, 1H), 6.75 (d, J=
9.0 Hz, 2H), 4.71 (s, 3H), 4.52 (d, J=5.9 Hz, 2H), 4.35 (d, J=2.0
Hz, 2H), 3.26 (t, J=2.0 Hz, 1H). HRMS [M þ H] calculated for
C23H20ClN3O 390.1375, found 390.1697.
4-[(2-Chlorobenzyl)(cyclopropylmethyl)amino]-N-(pyridin-3-
ylmethyl)benzamide (20h). 1H NMR (DMSO-d6) δ: 8.79 (t, J=
6.0 Hz, 1H), 8.68 (d, J=1.6 Hz, 1H), 8.63 (dd, J=1.2, 5.2 Hz,
1H), 8.08 (d, J=8.0 Hz, 1H), 7.71-7.67 (m, 3H), 7.48 (dd, J=
1.6, 7.6 Hz, 1H), 7.30-7.21 (m, 2H), 7.06 (dd, J=1.6, 7.6 Hz,
1H), 6.66 (d, J=8.8 Hz, 2H), 4.72 (s, 2H), 4.51 (d, J=5.6 Hz,
2H), 3.43 (d, J=6.4 Hz, 2H), 1.5-1.08 (m, 1H), 0.48-.043 (m,
2H), 0.28-.024 (m, 2H). HRMS [M þ H] calculated for
C24H24ClN3O 406.168 07, found 406.167 98.
4-[(2-Chlorobenzyl)(3-methylbut-2-en-1-yl)amino]-N-(pyridin-
3-ylmethyl)benzamide (20i). 1H NMR (DMSO-d6) δ: 8.73 (t, J=
6.0 Hz, 1H), 8.55 (d, J=1.2 Hz, 1H), 8.48 (dd, J=1.2, 3.6 Hz,
1H), 7.77 (dt, J=6.4, 2.0 Hz, 1H), 7.70 (d, J=8.8 Hz, 2H), 7.48
(dd, J = 1.6, 4.4 Hz, 1H), 7.42 (dd, J = 4.4, 6.4 Hz, 1H),
7.31-7.24 (m, 2H), 7.09 (dd, J=2.0, 6.4 Hz, 1H), 6.62 (d, J=
7.2 Hz, 2H), 5.26-5.23 (m, 1H), 4.61 (s, 2H), 4.45 (d, J=4.8 Hz,
2H), 4.07 (d, J=6.4 Hz, 2H), 1.69 (s, 3H), 1.65 (s, 3H). HRMS
[M þ H] calculated for C25H26ClN3O [M þ H] 420.183 72, found
420.185 09.
4-[(2E)-But-2-en-1-yl(2-chlorobenzyl)amino]-N-(pyridin-3-yl-
methyl)benzamide (20j). 1H NMR (DMSO-d6) δ: 8.79 (t, J=5.6
Hz, 1H), 8.67 (d, J=1.2 Hz, 1H), 8.62 (dd, 1.2, 5.2 Hz, 1H), 8.06
(d, J=7.6 Hz, 1H), 7.70 -7.64 (m, 3H), 7.50 (dd, J=2.0, 8.0 Hz,
1H), 7.32-7.25 (m, 2H), 7.05 (dd, J = 2.0, 7.2 Hz, 1H), 6.63
(d, J=9.6 Hz, 2H), 5.66-5.50 (m, 2H), 4.61 (s, 2H), 4.51 (d, J=
6.0 Hz, 2H), 4.05 (d, J=5.2 Hz, 2H), 1.65 (dd, J=1.2, 6.4 Hz,
3H). HRMS [M þ H] calculated for C24H24ClN3O 406.168 07,
found 406.168 44.
6-(Bromomethyl)-3-methylquinazolin-4-one (14a). Quinazoli-
nones 13a and 13b are brominated analogously to 11a.
3,6-Dimethyl-7-chloro-1,2,3-benzotriazin-4-one (15). DIEA
(3 equiv) is added to 8 (1 equiv), methylamine hydrochloride
(1.2 equiv), EDCI (1.2 equiv), and HOBt (1.2 equiv) in DCM
and the solution stirred at room temperature overnight. The
solution is concentrated and purified by MPLC (0-20% MeOH/
DCM). The resulting solid is suspended in 3.6 M HCl and
NaNO2/H2O (1.1 equiv, 2 M) is added dropwise at 0 °C. The
solution is warmed from 0 °C to room temperature over 2 h, and
3 mL of 10 N NaOH is added. Concentrated HCl is added to
obtain pH 2 and the solution cooled at 0 °C. The resulting solid is
filtered and dried under vacuum.
6-(Bromomethyl)-3-methyl-1,2,3-benzotriazin-4-one (16). Quin-
azolinone 15 is brominated analogously to 11.
General Procedure for the Synthesis of 20, 22, and 23. Alkyl
bromide (1.2 equiv) is added to ethyl 4-aminobenzoate or tert-
butyl 4-aminobenzoate (1 equiv) and 2,6-lutidine (1.2 equiv)
in DMF (0.5 M) and the solution heated at 70 °C overnight.
The solution is concentrated and purified by MPLC (0-60%
EtOAc/hex).
Bromide 12, 14, 16, or 18 (1 equiv) is added to N-alkylated
4-aminobenzoate ester (1 equiv) and 2,6-lutidine (1.2 equiv) in
DMF (0.5 M) and the solution heated at 70 °C overnight. The
solution is concentrated and purified by MPLC (0-60% EtOAc/
hex). Ethyl esters are cleaved by adding NaOH (2.5 equiv) in 1/1
EtOH/H2O and heating at reflux for 1-2 h. The solution is
cooled, acidified with 10% HCl, and washed with EtOAc. The
organic layer is dried with Na2SO4, filtered, and concentrated.
tert-Butyl esters are added to ∼5 mL/mmol 1:1 TFA/DCM and
stirred at room temperature for 30-90 min and concentrated.
The resulting acids are dissolved in DCM, and 3-aminomethyl-
pyridine (1.2 equiv), DIC (1.2 equiv), HOBt (1.2 equiv) are
added followed by DIEA (3 equiv). The solution is stirred at
room temperature overnight, concentrated, and purified by
MPLC (0-15% MeOH/DCM).
4-[(2-Chlorobenzyl)(methyl)amino]-N-(pyridin-3-ylmethyl)-
1
benzamide (20a). H NMR (DMSO-d6) δ: 8.69-8.62 (m, 2H),
8.12 (bs, 1H), 7.73 (t, J=6.0 Hz, 1H), 7.44-7.32 (m, 5H), 7.30
(d, J=9.0 Hz, 2H), 6.52 (d, J=9.0 Hz, 2H), 4.70 (s, 4H), 2.69 (s,
3H). HRMS [M þ H] calculated for C21H20ClN3O 366.136 77,
found 366.136 83.
4-[(2-Chlorobenzyl)(ethyl)amino]-N-(pyridin-3-ylmethyl)benzamide
(20b). 1H NMR (DMSO-d6) δ: 8.80 (t, J=5.6 Hz, 1H), 8.69 (d,
J=2.0 Hz, 1H), 8.63 (dd, J=1.2, 8.4 Hz, 1H), 8.09 d, J=8.0 Hz,
1H), 7.72-7.68 (m, 3H), 7.50 (dd, 1.6, 8.0 Hz, 1H), 7.32-7.24
(m, 2H), 7.03 (dd, J=2.0, 7.6 Hz, 1H), 6.62 (d, 9.6, 2H), 4.62 (s,
2H), 4.52 (d, J=5.6 Hz, 2H), 3.55 (q, 7.2 Hz, 2H), 1.17 (t, J=6.8
Hz, 3H). HRMS [M þ H] calculated for C22H22ClN3O
380.1524, found 380.149 92.
4-[(2-Chlorobenzyl)(propyl)amino]-N-(pyridin-3-ylmethyl)-
benzamide (20c). H NMR (DMSO-d6) δ: 8.72 (t, J = 6.4 Hz,
4-[Benzyl(2-chlorobenzyl)amino]-N-(pyridin-3-ylmethyl)benzamide
(20k). 1H NMR (DMSO-d6) δ: 8.77 (t, J=5.6 Hz, 1H), 8.65 (br s,
1H), 8.60 (d, J=4.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.68-7.62
(m, 3H), 7.50 (dd, J=2.4, 6.8 Hz, 1H), 7.36-7.23 (m, 7 H), 7.12
(dd, J=2.8, 6.4 Hz, 1H), 6.64 (d, J=9.2 Hz, 2H), 4.79 (d, J=6.8
Hz, 4H), 4.49 (d, J=6.0 Hz, 2H). HRMS [M þ H] calculated for
C27H24ClN3O 442.168 07, found 442.167 12.
N-(1H-Benzimidazol-2-ylmethyl)-4-{[(7-chloro-2,3-dimethyl-
4-oxo-3,4-dihydroquinazolin-6-yl)methyl](prop-2-yn-1-yl)amino}-
benzamide (22a). 1H NMR (DMSO-d6) δ: 7.87 (s, 1H), 7.82 (d,
J=9.0 Hz, 2H), 7.75 (s, 1H), 7.74-7.66 (m, 2H), 7.49-7.42 (m,
2H), 6.84 (d, J=9.0 Hz, 2H), 4.83 (d, J=5.1 Hz, 2H), 4.79 (s, 2H),
4.41 (d, J =2.0 Hz, 2H), 3.47 (s, 3H), 3.24 (t, J = 2.0 Hz, 1H),
1
1H), 8.53 (s, 1H), 8.46 (dd, J=1.2, 4.8 Hz, 1H), 7.73 (dt, J=8.0,
2.0 Hz, 1H), 7.69 (d, J=9.2 Hz, 2H), 7.50 (dd, J=1.6, 8.0 Hz,
1H), 7.38 (ddd, J=0.8, 4.0, 7.6 Hz, 1H), 7.31-7.22 (m, 2H), 7.00
(dd, J=2.0, 7.6 Hz, 1H), 6.60 (d, J=9.2 Hz, 2H), 4.65 (s, 2H),
4.45 (d, J=5.6 Hz, 2H), 3.44 (t, J=7.6 Hz, 2H), 1.68-1.58 (m,
2H), 0.91 (t, J = 7.2 Hz, 3H). HRMS [M þ H] calculated for
C23H24Cl N3O 394.168 07, found 394.171 49.
4-[Butyl(2-chlorobenzyl)amino]-N-(pyridin-3-ylmethyl)benz-
amide (20d). 1H NMR (DMSO-d6) δ: 8.73 (d, J=4.3 Hz, 1H),
8.70 (s, 1H), 7.85 (dd, J=7.8, 5.5 Hz, 1H), 7.42-7.30 (m, 5H),
7.27 (d, J=8.6 Hz, 2H), 6.54 (d, J=8.6 Hz, 2H), 4.72 (s, 4H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8743
2.56 (s, 3H). HRMS [M þ H] calculated for C29H25ClN6O2
525.180 03, found 525.178 71.
(qd, J=9.4, 2.3 Hz, 1H), 7.03 (td, J=7.8, 2.3 Hz, 1H), 6.83 (d,
J=9.0 Hz, 2H), 4.80 (s, 2H), 4.43 (d, J=5.5 Hz, 2H), 4.34 (d, J=
2.3 Hz, 2H), 3.57 (s, 3H), 3.24 (t, J=2.3 Hz, 1H), 2.57 (s, 3H).
HRMS [M þ H] calculated for C28H24F2N4O2 487.194 01,
found 487.192 88.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(prop-2-yn-1-yl)amino}-N-(2,4,5-trifluorobenzyl)benzamide (22k).
1H NMR (DMSO-d6) δ: 8.73 (t, J=5.9 Hz, 1H), 8.01 (d, J=1.6
Hz, 1H), 7.74 (d, J=9.0 Hz, 2H), 7.72 (dd, J=8.7, 2.0 Hz, 1H),
7.56 (d, J=8.2 Hz, 1H), 7.55-7.50 (m, 1H), 7.40-7.32 (m, 1H),
6.83 (d, J=9.3 Hz, 2H), 4.80 (s, 2H), 4.41 (d, J=5.5 Hz, 2H),
4.35 (d, J=2.4 Hz, 2H), 3.51 (s, 3H), 3.24 (t, J=2.4 Hz, 1H), 2.58
(s, 3H). HRMS [M þ H] calculated for C28H23F3N4 O2
505.184 59, found 505.184 11.
4-{[(7-Chloro-2,3-dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)-
methyl](prop-2-yn-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide
(22b). 1H NMR (DMSO-d6) δ: 8.88 (t, J=5.8 Hz, 1H), 8.66 (s,
1H), 8.59 (d, J=5.1 Hz, 1H), 8.01 (d, J=7.8 Hz, 1H), 7.88 (s,
1H), 7.76 (d, J=9.0 Hz, 2H), 7.74 (s, 1H), 7.62 (dd, J=7.8, 5.1
Hz, 1H), 6.79 (d, J=9.0 Hz, 2H), 4.77 (s, 2H), 4.52 (d, J=5.8 Hz,
2H), 4.38 (d, J=2.3 Hz, 2H), 3.46 (s, 3H), 3.24 (t, J=2.3 Hz,
1H), 2.56 (s, 3H). HRMS [M þ H] calculated for C27H24ClN5O2
486.169 13, found 486.167 90.
4-{[(7-Chloro-2,3-dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)-
methyl](3-methylbut-2-en-1-yl)amino}-N-(pyridin-3-ylmethyl)-
1
benzamide (22c). H NMR (DMSO-d6) δ: 8.76 (t, J = 5.8 Hz,
1H), 8.59 (d, J=1.5 Hz, 1H), 8.53 (dd, J=3.5, 1.5 Hz, 1H), 8.09
(s, 1H), 7.86 (d, J=7.8 Hz, 1H), 7.70 (d, J=9.0 Hz, 2H), 7.51 (s,
1H), 7.48 (d, J=8.5 Hz, 1H), 6.78 (d, J=9.0 Hz, 2H), 5.30 (t, J=
6.2 Hz, 1H), 4.78 (s, 2H), 4.48 (d, J=6.3 Hz, 2H), 4.07 (d, J=5.8
Hz, 2H), 3.56 (s, 3H), 2.44 (s, 3H), 1.67 (s, 3H), 1.63 (s, 3H).
HRMS [M þ H] calculated for C29H30ClN5O2 516.216 08,
found 516.215 77.
N-(1H-Benzimidazol-2-ylmethyl)-4-{[(7-chloro-2,3-dimethyl-
4-oxo-3,4-dihydroquinazolin-6-yl)methyl](3-methylbut-2-en-1-yl)-
amino}benzamide (22d). 1H NMR (DMSO-d6) δ: 12.13 (s, 1H),
8.79 (t, J=5.9 Hz,1H), 8.08 (s, 1H), 7.76 (d, J=9.0 Hz, 2H), 7.52
(s, 1H), 7.42 (d, J=6.6 Hz, 1H), 7.15-7.08 (m, 2H), 6.80 (d, J=
9.0 Hz, 2H), 5.31 (t, J=6.4 Hz, 1H), 4.79 (s, 2H), 4.64 (d, J=5.9
Hz, 2H), 4.09 (d, J=6.4 Hz, 2H), 3.57 (s, 3H), 2.45 (s, 3H), 1.67
(s, 3H), 1.63 (s, 3H). HRMS [M þ H] calculated for
C31H31ClN6O2 555.226 98, found 555.226 64.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(prop-2-yn-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide (22e).
1H NMR (DMSO-d6) δ: 8.77 (t, J=5.8 Hz, 1 h), 8.51 (d, J=
1.6 Hz, 1H), 8.43 (dd, J=5.0, 1.9 Hz, 1H), 8.00 (d, J=1.6 Hz,
1H), 7.73 (d, J=9.0 Hz, 2H), 7.69 (tm, J=7.6 Hz, 1H), 7.55 (d,
J=8.6 Hz, 1H), 7.33 (dd, J=7.8, 4.7 Hz, 1H), 6.84 (d, J=9.0 Hz,
2H), 4.78 (s, 2H), 4.45 (d, J=5.8 Hz, 2H), 4.33 (d, J=2.4 Hz,
2H), 3.51 (s, 3H), 3.22 (t, J=2.4 Hz, 1H), 2.56 (s, 3H). HRMS
[M þ H] calculated for C27H25N5O2 452.208 10, found
452.210 90.
N-(4-Chlorobenzyl)-4-{[(2,3-dimethyl-4-oxo-3,4-dihydroqui-
nazolin-6-yl)methyl](prop-2-yn-1-yl)amino}benzamide (22l). HRMS
[M þ H] calculated for C28H25ClN4O2 485.173 88, found 485.173 31.
N-(3,4-Difluorobenzyl)-4-{[(2,3-dimethyl-4-oxo-3,4-dihydro-
quinazolin-6-yl)methyl](prop-2-yn-1-yl)amino}benzamide (22m).
1H NMR (DMSO-d6) δ: 8.77 (t, J=6.2 Hz, 1H), 8.00 (d, J=2.0
Hz, 1H), 7.74 (d, J=9.4 Hz, 2H), 7.71 (dd, J=8.2, 2.0 Hz, 1H),
7.56 (d, J=8.2 Hz, 1H), 7.38-7.27 (m, 2H), 7.14-7.09 (m, 1H),
6.84 (d, J=9.0 Hz, 2H), 4.79 (s, 2H), 4.41 (d, J=6.2 Hz, 2H),
4.34 (d, J=2.3 Hz, 2H), 3.51 (s, 3H), 3.24 (t, J=2.3 Hz, 1H), 2.57
(s, 3H). HRMS [M þ H] calculated for C28H24F2N4O2 487.194 01,
found 487.194 85.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(prop-2-yn-1-yl)amino}-N-(3-fluorobenzyl)benzamide (22n). 1H
NMR (DMSO-d6) δ: 8.77 (t, J=6.2 Hz, 1H), 8.00 (d, J=2.0
Hz, 1H), 7.75 (d, J=9.0 Hz, 2H), 7.70 (dd, J=6.6, 2.0 Hz, 1H),
7.56 (d, J=8.2 Hz, 1H), 7.38-7.31 (m, 1H), 7.14-7.01 (m, 3H),
6.84 (d, J=9.4 Hz, 2H), 4.79 (s, 2H), 4.44 (d, J=5.9 Hz, 2H),
4.34 (d, J=2.4 Hz, 2H), 3.51 (s, 3H), 3.24 (t, J=2.0, 1H), 2.56 (s,
3H). HRMS [M þ H] calculated for C28H25FN4O2 469.203 43,
found 469.202 76.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(3-methylbut-2-en-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide
(22o). 1H NMR (DMSO-d6) δ: 8.80 (t, J=5.2 Hz, 1H), 8.70 (d,
J=2.8 Hz, 1H), 8.65 (d, J=2.5 Hz, 1H), 8.12 (bs, 1H), 7.91 (d,
J=1.6 Hz, 1H), 7.69 (d, J=9.4 Hz, 2H), 7.66 (dd, J=8.2, 2.3 Hz,
1H), 7.56 (d, J=8.6 Hz, 1H), 6.71 (d, J=9.0 Hz, 2H), 5.22 (t, J=
6.2 Hz, 1H), 4.73 (s, 2H), 4.52 (d, J=5.5 Hz, 2H), 4.10 (d, J=6.8
Hz, 2H), 3.51 (s, 3H), 2.57 (s, 3H), 1.68 (s, 3H), 1.66 (s, 3H).
HRMS [M þ H] calculated for C29H31N5O2 482.255 05, found
482.257 55.
N-(1H-Benzimidazol-2-ylmethyl)-4-{[(2,3-dimethyl-4-oxo-3,4-
dihydroquinazolin-6-yl)methyl](prop-2-yn-1-yl)amino}benzamide
(22f). HRMS [M þ H] calculated for C29H26N6O2 491.2190,
found 491.2182.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(prop-2-yn-1-yl)amino}-N-(piperidin-3-ylmethyl)benzamide (22g).
HRMS [M þ H] calculated for C27H31N5O2 458.255 05, found
458.254 45.
N-(1H-Benzimidazol-2-ylmethyl)-4-{[(2,3-dimethyl-4-oxo-3,4-
dihydroquinazolin-6-yl)methyl](3-methylbut-2-en-1-yl)amino}-
1
benzamide (22p). H NMR (DMSO-d6) δ: 9.04 (t, J = 5.1 Hz,
N-(1,3-Benzodioxol-5-ylmethyl)-4-{[(2,3-dimethyl-4-oxo-3,4-
dihydroquinazolin-6-yl)methyl](prop-2-yn-1-yl)amino}benzamide
(22h). 1H NMR (DMSO-d6) δ: 8.66 (t, J=6.0 Hz, 1H), 8.00 (d,
J=2.0 Hz, 1H), 7.73 (d, J=9.0 Hz, 2H), 7.70 (dd, J=8.6, 2.3 Hz,
1H), 7.55 (d, J=8.2 Hz, 1H), 6.85-6.81 (m, 4H), 6.75 (dd, J=
7.8, 1.5 Hz, 1H), 5.99 (s, 2H), 4.79 (s, 2H), 4.36-4.30 (m, 4H),
3.51 (s, 3H), 3.23 (t, J=2.0, 1H), 2.56 (s, 3H). HRMS [M þ H]
calculated for C29H26N4O4 495.202 68, found 495.202 14.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
1H), 7.90 (d, J=1.5 Hz, 1H), 7.77-7.70 (m, 4H), 7.65 (dd, J=
8.6, 1.9 Hz, 1H), 7.57 (d, J=8.6 Hz, 1H), 7.52-7.47 (m, 2H),
6.76 (d, J=9.4 Hz, 2H), 5.23 (t, J=6.6 Hz, 1H), 4.84 (d, J=5.1
Hz, 2H), 4.76 (s, 2H), 4.12 (d, J=6.6 Hz, 2H), 3.51 (s, 3H), 2.56
(s, 3H), 1.69 (s, 3H), 1.67 (s, 3H). HRMS [M þ H] calculated for
C31H32N6O2 521.265 95, found 521.266 21.
4-{[(7-Chloro-3-methyl-4-oxo-3,4-dihydro-1,2,3-benzotriazin-
6-yl)methyl](3-methylbut-2-en-1-yl)amino}-N-(pyridin-3-ylmethyl)-
1
benzamide (22q). H NMR (DMSO-d6) δ: 8.82 (t, J = 5.9 Hz,
1
(prop-2-yn-1-yl)amino}-N-(2-fluorobenzyl)benzamide (22i). H
1H), 8.65 (d, J=2.1 Hz, 1H), 8.59 (dd, J=4.7, 2.1 Hz, 1H), 8.43
(s, 1H), 8.04-7.98 (m, 1H), 7.86 (s, 1H), 7.72 (d, J=9.0 Hz, 2H),
7.63 (bs, 1H), 6.69 (d, J=9.4 Hz, 2H), 5.28 (t, J=6.6 Hz, 1H),
4.79 (d, J=5.9 Hz, 2H), 4.51 (d, J=5.9 Hz, 2H), 4.15 (d, J=6.6
Hz, 2H), 3.87 (s, 3H), 1.69 (s, 3H), 1.66 (s, 3H). HRMS [M þ H]
calculated for C27H27ClN6O2 503.195 68, found 503.196 89.
4-{[(3-Methyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl](prop-2-
yn-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide (23a). 1H NMR
(DMSO-d6) δ: 8.91 (t, J=5.9 Hz, 1H), 8.79 (d, J=1.6 Hz, 1H),
8.74 (dd, J=4.3, 1.6 Hz, 1H), 8.37 (s, 1H), 8.31 (d, J=8.2 Hz,
1H), 8.05 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 7.8, 5.4 Hz, 1H),
7.77-7.72 (m, 3H), 7.65 (d, J=8.2 Hz, 1H), 6.84 (d, J=9.0 Hz,
2H), 4.82 (s, 2H), 4.57 (d, J=5.8Hz, 2H), 4.36 (d, J=2.0Hz, 2H),
NMR (DMSO-d6) δ: 8.71 (t, J=6.0 Hz, 1H), 8.01 (d, J=2.0
Hz, 1H), 7.75 (d, J=9.0 Hz, 2H), 7.72 (dd, J=8.6, 1.6 Hz, 1H),
7.57 (d, J=8.2 Hz, 1H), 7.34-7.25 (m, 2H), 7.15 (q, J=7.4 Hz,
2H), 6.84 (d, J=9.0 Hz, 2H), 4.80 (s, 2H), 4.47 (d, J=5.8 Hz,
2H), 4.35 (d, J=2.0 Hz, 2H), 3.52 (s, 3H), 3.24 (t, J=2.0 Hz,
1H), 2.58 (s, 3H). HRMS [M þ H] calculated for C28H25FN4O2
469.203 43, found 429.202 11.
N-(2,4-Difluorobenzyl)-4-{[(2,3-dimethyl-4-oxo-3,4-dihydro-
quinazolin-6-yl)methyl](prop-2-yn-1-yl)amino}benzamide (22j).
1H NMR (DMSO-d6) δ: 8.71 (t, J=5.9 Hz, 1H), 8.00 (d, J=
1.6 Hz, 1H), 7.74 (d, J=9.0 Hz, 2H), 7.70 (dd, J=8.6, 2.0 Hz,
1H), 7.56 (d, J = 8.6 Hz, 1H), 7.35 (q, J = 6.6 Hz, 1H), 7.21

8744 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Lockman et al.
3.47 (s, 3H), 3.25 (t, J=2.0 Hz, 1H). HRMS [M þ H] calculated
for C26H23N5O2 438.19245, found 438.19223.
2.57 (s, 3H), 1.66 (d, J=6 Hz, 3H). HRMS [M þ H] calculated
for C28H29N5O2 468.2394, found 468.240 15.
4-{(3-Methylbut-2-en-1-yl)[(3-methyl-4-oxo-3,4-dihydroquin-
azolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide (23b).
1H NMR (DMSO-d6) δ: 8.81 (t, J=6.8 Hz, 1H), 8.72 (d, J=1.8
Hz, 1H), 8.67 (d, J=5.1, 1.8 Hz, 1H), 8.36 (s, 1H), 8.18 (d, J=8.6
Hz, 1H), 7.97 (d, J=1.2 Hz, 1H), 7.80-7.75 (m, 1H), 7.72-7.62
(m, 4H), 6.71 (d, J=9.4 Hz, 2H), 5.23 (t, J=6.5 Hz, 1H), 4.75 (s,
2H), 4.53 (d, J=5.8 Hz, 2H), 4.11 (d, J=6.8 Hz, 2H), 3.47 (s,
3H), 1.69 (s, 3H), 1.67 (s, 3H). HRMS [M þ H] calculated for
C28H29N5O2 468.239 40, found 468.239 86.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(3-methylbut-2-en-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide
(23j). H NMR (DMSO-d6) δ: 9.20 (s, 1H), 9.17 (d, J=6 Hz,
1H), 8.85 (m, 1H), 8.51 (d, J=8 Hz, 1H), 8.35 (s, 1H), 8.15 (dd,
J=7, 6, 6 Hz, 1H), 7.93 (dd, J=8, 2, 2 Hz, 1H), 7.68-7.56 (m,
3H), 6.56 (d, J=9 Hz, 2H), 5.99 (s, 2H), 5.24 (m, 1H), 4.59 (d, J=
5 Hz, 2H), 3.66 (d, J=6 Hz, 1H), 3.54 (s, 3H), 2.60 (s, 3H), 2.0 (s,
3H), 1.70 (d, J = 5 Hz, 3H). HRMS [M þ H] calculated for
C29H31N5O2 482.255 05, found 482.261 05.
1
4-{[(7-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(3-methylbut-2-en-1-yl)amino}-N-(pyridin-3-ylmethyl)benzamide
(23c). 1H NMR (DMSO-d6) δ: 8.80 (t, J=5.8 Hz, 1H), 8.64 (d,
J=1.5 Hz, 1H), 8.58 (dd, J=5.1 Hz, 1.4, 1H), 8.39 (s, 1H), 7.98
(d, J=7.8 Hz, 1H), 7.84 (d, J=3.1 Hz, 2H), 7.72 (d, J=9.0 Hz,
2H), 7.60 (dd, J=7.9, 5.1 Hz, 1H), 6.67 (d, J=9.0 Hz, 2H), 5.27
(t, J=6.2 Hz, 1H), 4.71 (s, 2H), 4.50 (d, J=5.5 Hz, 2H), 4.13 (d,
J=6.2 Hz, 2H), 3.43 (s, 3H), 1.69 (s, 3H), 1.67 (s, 3H). HRMS
[Mþ H] calculated for C28H28ClN5O2 502.200 43, found 502.202 60.
4-{[(7-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(cyclopropylmethyl)amino}-N-(pyridin-3-ylmethyl)benzamide (23d).
1H NMR (DMSO-d6) δ: 8.78 (dd, J=6, 6 Hz, 1H), 8.59 (s, 1H),
8.52 (d, J=4 Hz, 1H), 8.38 (s, 1H), 7.86 (s, 1H), 7.84 (s, 1H), 7.71
(d, J=9 Hz, 2H), 7.49 (dd, J=7, 5, 5 Hz, 1H), 6.71 (d, J=8 Hz,
2H), 4.82 (s, 2H), 4.47 (d, J=5 Hz, 2H), 3.49 (d, J=6 Hz, 2H),
3.41 (s, 3H), 1.20-1.08 (m, 1H), 0.48-0.42 (m, 2H), 0.30-0.25
(m, 2H). HRMS [M þ H] calculated for C27H26ClN5O2
488.1847, found 488.182 95.
4-{(2-Amino-2-oxoethyl)[(7-chloro-3-methyl-4-oxo-3,4-dihydro-
quinazolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide
(23e). 1H NMR (DMSO-d6) δ: 8.87 (dd, J=5, 5 Hz, 1H), 8.72 (s,
1H), 8.67 (d, J=5 Hz, 1H), 8.40 (s, 1H), 8.16 (d, J=7 Hz, 1H),
7.85 (s, 1H), 7.79-7.73 (m, 2H), 7.71 (s, 1H), 7.57 (s, 1H), 7.24 (s,
1H), 6.58 (d, J=8 Hz, 2H), 4.82 (s, 2H), 4.54 (d, J=5 Hz, 2H),
4.13 (s, 2H), 3.42 (s, 3H). HRMS [M þ H] calculated for
C25H23ClN6O3 491.159 29, found 491.159 06.
4-{But-3-yn-1-yl[(7-chloro-3-methyl-4-oxo-3,4-dihydroquin-
azolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide (23f).
1H NMR (DMSO-d6) δ: 8.86 (dd, J = 5, 5 Hz, 1H), 8.70 (s,
1H), 8.65 (d, J=4 Hz, 1H), 8.39 (s, 1H), 8.12 (d, J=8 Hz, 1H),
7.85 (s, 1H), 7.73 (d, J=4 Hz, 2 Hz), 7.71 (s, 1H), 6.71 (d, J=9
Hz, 2H), 4.83 (s, 2H), 4.53 (d, J=5 Hz, 2H), 3.75 (dd, J=6, 6 Hz,
2H), 3.42 (s, 3H), 2.92 (d, J=2, 2 Hz, 1H), 2.59-2.53 (m, 2H).
HRMS [M þ H] calculated for C27H24ClN5O2 486.169 13,
found 486.1685.
4-{[(7-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(3-methylbut-2-enoyl)amino}-N-(pyridin-3-ylmethyl)benzamide
(23g). 1H NMR (DMSO-d6) δ: 9.21 (t, J=5.9 Hz, 1H), 8.74 (s,
1H), 8.66 (d, J=4.3 Hz, 1H), 8.39 (s, 1H), 8.16 (d, J=7.4 Hz,
1H), 8.07 (s, 1H), 7.85 (d, J=8.6 Hz, 2H), 7.73 (s, 1H), 7.33 (d,
J=8.6 Hz, 2H), 5.57 (s, 1H), 5.18 (s, 2H), 4.55 (d, J=5.5 Hz,
2H), 3.46 (s, 3H), 2.07 (s, 3H), 1.70 (s, 3H). HRMS [M þ H]
calculated for C28H26ClN5O3 516.179 69, found 516.182 69.
4-{[(3,5-Dimethyl-1,2-oxazol-4-yl)methyl][(2,3-dimethyl-4-oxo-
3,4-dihydroquinazolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)-
benzamide (23h). 1H NMR (DMSO-d6) δ: 8.88 (dd, J=6, 6 Hz,
1H), 8.77 (s, 1H), 8.72 (d, J=5 Hz, 1H), 8.27 (d, J=7 Hz, 1H),
7.88-7.81 (m, 2H), 7.71 (d, J=9Hz, 1H), 7.63 (dd, J=8, 1, 1 Hz,
1H), 7.56 (d, J=8 Hz, 1H), 6.85 (d, J=8 Hz, 2H), 4.77 (s, 2H),
4.58 (s, 2H), 4.55 (d, J=5 Hz, 2H), 3.51 (s, 3H), 2.58 (s, 3H), 2.24
(s, 3H), 2.12 (s, 3H). HRMS [M þ H] calculated for C30H30N6O3
524.229 23, found 524.249 44.
4-{(Cyclopropylmethyl)[(2,3-dimethyl-4-oxo-3,4-dihydroquin-
azolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide (23k).
1H NMR (DMSO-d6) δ: 8.79 (dd, J = 5, 5 Hz, 1H), 8.70 (s,
1H), 8.65 (d, J=5 Hz, 1H), 8.13 (d, J=1 Hz, 1H), 7.92 (d, J=1
Hz, 1H), 7.79-7.59 (m, 5H), 7.55 (d, J=8 Hz, 1H), 6.75 (d, J=8
Hz, 2H), 4.84 (s, 2H), 4.52 (d, J=5 Hz, 2H), 3.50 (s, 3H), 3.46 (d,
J = 6 Hz, 2 Hz), 1.19-1.08 (m, 1H), 0.49-0.42 (m, 2H),
0.31-0.25 (m, 2H). HRMS [M
þ
H] calculated for
C28H29N5O2 468.2394, found 468.2393.
4-{(Cyclopropylcarbonyl)[(2,3-dimethyl-4-oxo-3,4-dihydro-
quinazolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide
(23l). 1H NMR (DMSO-d6) δ: 9.22 (dd, J=5, 5 Hz, 1H), 8.75 (s,
1H), 8.68 (d, J=4 Hz, 1H), 8.18 (d, J=7 Hz, 7.91-7.84 (m, 3H),
7.76 (dd, J=7, 5, 5 Hz, 1H), 7.61 (dd, J=8, 1, 1 Hz, 1H), 5.09 (s,
1H), 4.57 (d, J=6, 2H), 3.50 (s, J=2H), 2.57 (s, 3H), 1.41 (bs,
1H), 0.97-0.88 (m, 2H), 0.75-0.66 (m, 2H). HRMS [M þ H]
calculated for C28H27N5O3 482.218 67, found 482.218 67.
4-{[(2,3-Dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl]-
(3-methylbut-2-enoyl)amino}-N-(pyridin-3-ylmethyl)benzamide
(23m). 1H NMR (DMSO-d6) δ: 9.24 (dd, J = 5, 5 Hz, 1H),
8.97-8.56 (m, 2H), 8.26 (d, J=7 Hz, 1H), 7.90 (d, J=2 Hz, 1H),
7.87-7.79 (m, 3H), 7.65 (dd, J=8, 2, 2 Hz, 1H), 7.53 (d, J=8 Hz,
2H), 5.53 (s, 1H), 5.11 (s, 2H), 4.58 (d, J=5 Hz, 2H), 3.51 (s, 3H),
2.58 (s, 3H), 2.08 (s, 3H), 1.68 (s, 3H). HRMS [M þ H]
calculated for C29H29N5O3 496.234 32, found 496.234 56.
4-(Prop-2-ynylamino)-N-(3-pyridylmethyl)benzamide (24). Pro-
pargyl bromide is added to ethyl 4-aminobenzoate (1.2 equiv) and
2,6-lutidine in DMF. The solution is heated at 70 °C overnight,
concentrated, and purified by MPLC (0-30% EtOAc/hex). The
resulting solid is dissolved in ethanol, and 2 M NaOH (2 equiv) is
added. The mixture is heated at reflux for 2.5 h. The ethanol is
removed under vacuum, ethyl acetate is added, and the organic
layer is washed with 10% HCl, dried with Na2SO4, and concen-
trated. DCM is added followed by 3-aminomethylpyridine, DIC
(1.2 equiv), HOBt (1.2 equiv), and DIEA (3 equiv). The solution is
stirred at room temperature overnight, concentrated, and purified
by MPLC (0-20% MeOH/DCM).
4-[{[7-Chloro-2-(hydroxymethyl)-3-methyl-4-oxo-3,4-dihy-
droquinazolin-6-yl]methyl}(prop-2-yn-1-yl)amino]-N-(pyridin-3-
ylmethyl)benzamide (26).1H NMR (DMSO-d6) δ:8.90(t,J=5.7 Hz,
1H), 8.71 (s, 1H), 8.65 (d, J=2.2 Hz, J=1H), 8.13 (d, J=4.3 Hz,
1H), 7.91 (s, 1H), 7.83 (s, 1H), 7.80-7.69 (m, 3H), 6.80 (d, J=4.3 Hz,
2H), 4.79 (s, 2H), 4.58 (s, 2H), 4.54 (d, J=2.7 Hz, 2H), 4.39 (s, 2H),
3.50 (s, 3H), 3.24 (d, J=2.0 Hz, 1H). HRMS [M þ H] calculated for
C27H24ClN5O3 502.1648, found 502.1687.
7-Chloro-3-methyl-4-oxoquinazoline-6-carboxylic Acid (28).
Quinazolinone 13 is dissolved in water/pyridine (2:1; 0.3 M)
and heated to 100 °C. Potassium permanganate (7 equiv) is
added in portions at 100 °C, and the solution is heated at reflux
for 5 h. and filtered over Celite. The filtrate is dilluted with
EtOAc, washed with 10% HCl, dried with Na2SO4, and con-
centrated. The acid is used crude.
4-{(2E)-But-2-en-1-yl[(2,3-dimethyl-4-oxo-3,4-dihydroquin-
azolin-6-yl)methyl]amino}-N-(pyridin-3-ylmethyl)benzamide (23i).
1H NMR (DMSO-d6) δ: 8.81 (dd, J=5, 5 Hz, 1H), 8.71 (s, 1H),
8.66 (d, J=5 Hz, 1H), 8.15 (d, J=7 Hz, 1H), 7.9 (d, J=1 Hz,
1H), 7.75 (dd, J=8, 5, 5 Hz, 1H), 7.72-7.64 (m, 3H), 7.57 (d, J=
8 Hz, 1H), 6.72 (d, J=9 Hz, 2H), 5.69-5.57 (m, 1H), 4.73 (s,
2H), 4.53 (d, J=5 Hz, 2H), 4.08 (d, J=4 Hz, 2H), 3.51 (s, 3H),
7-Chloro-3-methyl-4-oxo-N-[4-(3-pyridylmethylcarbamoyl)-
phenyl]quinazoline-6-carboxamide (29). TEPP (1.2 equiv) isadded
to acid 28 and 4-amino-N-(3-pyridylmethyl)benzamidein toluene
and the solution heated at reflux overnight. The solution was
concentrated and purified by RP-HPLC. 1H NMR (DMSO-d6)
δ: 10.92 (s, 1H), 9.12 (t, J=5.9 Hz, 1H), 8.68 (d, J=1.6 Hz, 1H),
8.59 (dd, J = 4.6, 1.6 Hz, 1H), 8.52 (s, 1H), 8.30 (s, 1H), 8.00

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8745
(d, J=7.7 Hz, 1H), 7.92 (d, J=9.0 Hz, 2H), 7.91 (s, 1H), 7.82 (d,
J=8.6 Hz, 2H), 7.60 (dd, J=8.5, 5.0 Hz, 1H), 4.56 (d, J=5.9 Hz,
2H), 3.53 (s, 3H). HRMS [M þ H] calculated for C23H18ClN5O3
448.117 09, found 448.12338.
6-Amino-7-chloro-3-methylquinazolin-4-one (30). DPPA and
Et3N are added to acid 28 in t-BuOH, and the solution is heated
at reflux overnight. Ethyl acetate is added and washed with satu-
rated NaHCO3, dried with Na2SO4, and concentrated. DCM
followed by TFA is added and the solution stirred at room
temperature for 90 min and then concentrated. Amine 30 is used
crude.
4-[[(7-Chloro-3-methyl-4-oxo-quinazolin-6-yl)amino]methyl]-
N-(3-pyridylmethyl)benzamide (31). TFA (2 equiv) is added to
amine 30 and 4-formylbenzoic acid in isopropyl acetate and the
solution stirred at room temperature for 10 min. NaBH(OAc)3
(1.5 equiv) is added and the mixture heated at reflux overnight,
concentrated, and purified by MPLC (0-20% MeOH/DCM).
The resulting acid is added to 3-aminomethylpyridine (1.2 equiv),
DIC (1.2 equiv), HOBt (1.2 equiv) in DMF. DIEA (3 equiv) is
added and the solution stirred overnight at room temperature,
concentrated, and purified by RP-HPLC. 1H NMR (DMSO-d6)
δ: 9.15 (t, J=5.5 Hz, 1H), 8.75 (d, J=1.5 Hz, 1H), 8.68 (dd, J=
4.3, 1.5 Hz, 1H), 8.20 (d, J=7.4 Hz, 1H), 8.12 (s, 1H), 7.84 (d, J=
8.2 Hz, 2H), 7.77 (dd, J=7.4, 5.5 Hz, 1H), 7.68 (s, 1H), 7.45 (d,
J=8.6 Hz, 2H), 6.98 (s, 1H), 6.86 (bs, 1H), 4.58 (m, 4H), 3.38 (s,
3H). HRMS [M þ H] calculated for C23H20ClN5O2 434.137 83,
found 434.138 57.
(m, 3H), 7.94 (d, J=7.4 Hz, 1H), 7.71 (d, J=8.6 Hz, 2H), 7.56
(dd, J=7.8, 5.0 Hz, 1H), 7.31 (bs, 2H), 7.09 (d, J=8.6 Hz, 2H),
3.52 (q, J=7.0 Hz, 2H), 2.92 (t, J=7.0 Hz, 2H), 2.35 (s, 3H).
HRMS [M þ H] calculated for C21H21N7OS 420.160 11, found
420.155 15.
4-[(3-Chloro-2-oxopropyl)prop-2-ynylamino]-N-(3-pyridyl-
methyl)benzamide (39). tert-Butyl bromoacetate is added to 24
and 2,6-lutidine in DMF, and the solution is heated at 70 °C
overnight, concentrated, and purified by MPLC (0-20%
MeOH/DCM). The resulting oil is dissolved in DCM, TFA is
added, and the mixture is stirred for 2 h and then concentrated.
The acid is then dissolved in DCM. Oxalyl chloride (2 equiv) is
added followed by 0.5 mL of DMF. THF is added after 5 min to
aid in solubility. The solution is stirred for 1 h and then con-
centrated. THF is followed by CH2N2 (2 M in hexanes, 2 equiv).
The mixture is stirred at room temperature for 4 h, then cooled
to 0 °C and HCl (g) is bubbled through the solution for 10 min.
The resulting solid is filtered, dried under vacuum, and carried
on crude.
4-[(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)methylprop-2-ynyl-
amino]-N-(3-pyridylmethyl)benzamide (40). 2,6-Diamino-3,4-di-
hydropyrim-4-one is added to chloride 39 in DMF and the
solution heated at 70 °C overnight and 150 °C for an additional
4 h. The solution is filtered, washed with a small amount of
MeOH, and the solid is purified by RP-HPLC. 1H NMR
(DMSO-d6) δ: 8.94 (s, 1H), 8.93-8.81 (m, 2H), 8.46 (d, J =
8.2 Hz, 1H), 8.08 (dd, J=8.2, 6.3 Hz, 1H), 7.71 (d, J=8.6 Hz,
2H), 6.67 (d, J=9.0 Hz, 2H), 4.59 (d, J=5.8 Hz, 2H), 4.35 (s,
2H), 3.94 (d, J=2.4 Hz, 2H), 3.12 (t, J=2.4 Hz, 1H). HRMS
[M þ H] calculated for C23H21N7O2 427.175 67.
2-Amino-6-methyl-3,7-dihydropyrrolo[2,3-d]pyrimidin-4-one
(32)/5-Methylfuro[2,3-d]pyrimidine-2,4-diamine14b (33). Chlor-
acetone is added to 2,6-diamino-3,4-dihydropyrim-4-one in
DMF and the solution heated at 50 °C overnight, concentrated,
and purified by MPLC (0-20% MeOH/DCM). Fraction 1
contains 32. Fraction 2 contains 33.
4-[2-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic
Acid (41). Et3N (20 mol %) is added to ethyl 4-(3-oxopropyl)-
benzoate, paraformaldehyde, and 3-ethylthiazoline bromide in
EtOH. The solution is heated at reflux overnight, concentrated,
and purified by MPLC (0-100% EtOAc/hex). The resulting
hydroxyketone is added to malononitrile and Et3N in MeOH
and the solution stirred at room temperature overnight and
filtered. The solid is added to a premixed (5 min) solution of
guanidine hydrochloride (1.5 equiv) and KOEt (1.5 equiv) in
EtOH and the mixture heated at reflux overnight. The mixture
was concentrated and the resulting acid carried on crude.
4-[2-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
N-(3-pyridylmethyl)benzamide (42). DMF, 3-aminomethylpyr-
idine (1.2 equiv), DIC (1.2 equiv), HOBt (1.2 equiv), and DIEA
(3 equiv) are added to acid 41. The solution is stirred at room
temperature overnight, concentrated, and purified by RP-HPLC.
1H NMR (DMSO-d6) δ: 11.48 (s, 1H), 9.07 (t, J=5.8 Hz, 1H),
8.58 (d, J=1.6 Hz, 1H), 8.50 (dd, J=4.7, 1.7 Hz, 1H), 7.81 (d,
J=8.2 Hz, 2H), 7.79 (bs, 1H), 7.44 (dd, J=7.8, 5.1 Hz, 1H), 7.35
(d, J = 8.2 Hz, 2H), 6.59 (bs, 2H), 4.51 (d, J = 5.9 Hz, 2H),
3.06-2.98 (m, 2H), 2.96-2.90 (m, 2H). HRMS [M þ H] calc-
ulated for C21H21N7O 388.188 03, found 388.178 32.
2-Methyl-5,6,7,8-tetrahydro-3H-pyrido[4,3-d]pyrimidin-4-one
(43). To a round bottomed flask methyl 4-oxopiperidine-3-
carboxylate (1.0 equiv) and acetamidine HCl (1.1 equiv) were
added with stirring in EtOH at room temperature. While the
mixture was being stirred, Na (2.1 equiv) was dissolved in
another flask containing EtOH. Upon dissolution, the above
mixture was added to the mixture containing the acetamidine
mixture, and the mixture was heated at 100 °C overnight. The
reaction mixture was removed from heat, and upon cooling the
mixture was filtered to remove solids, then concentrated to yield
2-methyl-5,6,7,8-tetrahydro-3H-pyrido[4,3-d]pyrimidin-4-one as a
cream colored solid.
4-[(2-Amino-6-methyl-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-
5-yl)sulfanyl]benzoic Acid (34). Heterocycle 32, 4-mercaptoben-
zoic acid (1.75 equiv), is heated at reflux in 2:1 ethanol/water for
5 min. Iodine (1.75 equiv) is added and the solution heated at
reflux for 3 h, cooled, and filtered. A solution of 10% Na2S2O3 is
added to the filtrate and the mixture stirred at room temperature
overnight, filtered, concentrated, and carried on crude.
4-[(2-Amino-6-methyl-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-
5-yl)sulfanyl]-N-(3-pyridylmethyl)benzamide (35). 3-Aminomet-
hylpyridine (1.2 equiv), DIC (1.2 equiv), HOBt (1.2 equiv) are
added to 34 followed by DIEA (3 equiv). The solution is stirred
at room temperature overnight, concentrated, and purified by
1
RP-HPLC. H NMR (DMSO-d6) δ: 9.12 (t, J = 5.8 Hz, 1H),
8.61 (d, J=2.0 Hz, 1H), 8.54 (dd, J=4.6, 2.0 Hz, 1H), 7.88 (d,
J=7.0 Hz, 1H), 7.82 (d, J=9.0 Hz, 2H), 7.54-7.48 (m, 1H), 7.20
(d, J=8.6 Hz, 2H), 4.51 (d, J=5.5 Hz, 2H), 2.33 (s, 3H). HRMS
[Mþ H] calculated for C20H18N6O2S 407.128 47, found 407.125 27.
5-Methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (36). Et3N
is added to malononitrile and hydroxyacetone in MeOH and the
solution stirred at room temperature overnight and concen-
trated. The resulting solid is dissolved in ethanol and added to a
premixed solution of guanidine hydrochloride (1.5 equiv) and
NaOEt (1.5 equiv) in EtOH. The reaction mixture is heated at
reflux overnight, concentrated, and purified by MPLC (0-20%
MeOH/DCM).
4-[(2,4-Diamino-6-methyl-1H-pyrrolo[2,3-d]pyrimidin-5-yl)-
sulfanyl]-N-(pyridin-3-ylmethyl)benzamide (37). Addition of
4-mercaptobenzoic acid and subsequent amide coupling pro-
ceed as described for 35-36. 1H NMR (DMSO-d6) δ: 9.10 (t,
J=5.9 Hz, 1H), 8.59 (s, 1H), 8.53 (d, J=4.0 Hz, 1H), 7.81 (d,
J=8.6 Hz, 2H), 7.48 (t, J=5.0 Hz, 1H), 7.30 (bs, 1H), 7.12 (d,
J=8.6 Hz, 2H), 4.50 (d, J=6.3 Hz, 2H), 2.34 (s, 3H). HRMS
[M þ H] calculated for C20H19N7OS 406.144 46, found 406.133 72.
4-[(2,4-Diamino-6-methyl-1H-pyrrolo[2,3-d]pyrimidin-5-yl)-
sulfanyl]-N-[2-(pyridin-3-yl)ethyl]benzamide (38). Addition of
4-mercaptobenzoic acid and subsequent amide coupling pro-
ceed as described for 35-36. 1H NMR (DMSO-d6) δ: 8.60-8.51
2-Methyl-4-oxo-N-[4-(3-pyridylmethylcarbamoyl)phenyl]-
3,5,7,8-tetrahydropyrido[4,3-d]pyrimidine-6-carboxamide (44). To a
round bottomed flask 43 (1.0 equiv) was added with stirring in
DCM at room temperature. To this solution ethyl 4-isocyanato-
benzoate (1.1 equiv) was slowly added. As the reaction proceeded,
solid crashed out of solution. After being stirred for 1.5 h, the

8746 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Lockman et al.
reaction mixture was filtered and the isolated precipitate was rinsed
with DCM and dried to yield ethyl 4-[(2-methyl-4-oxo-3,5,7,8-
tetrahydropyrido[4,3-d]pyrimidine-6-carbonyl)amino]benzoate as
a white solid.
(4) Fleischer, T. C.; Murphy, B. R.; Flick, J. S.; Terry-Lorenzo, R. T.;
Gao, Z.-H.; Davis, T.; McKinnon, R.; Ostanin, K.; Willardsen,
J. A.; Boniface, J. J. Chemical Proteomics Identifies Nampt as the
Target of CB30865, an Orphan Cytotoxic Compound. Chem. Biol.
2010, 17, 659–664.
To a round-bottomed flask ethyl 4-[(2-methyl-4-oxo-3,5,7,8-
tetrahydropyrido[4,3-d]pyrimidine-6-carbonyl)amino]benzoate
(1.0 equiv) was added with stirring in 1 N LiOH (5-10 equiv) at
room temperature overnight. After the reaction was determined
to be complete, the mixture was neutralized with HCl, at which
point a white solid crashed out of solution. The solid was filtered
and collected to yield 4-[(2-methyl-4-oxo-3,5,7,8-tetrahydropyrido-
[4,3-d]pyrimidine-6-carbonyl)amino]benzoic acid.
To a medium vial 4-[(2-methyl-4-oxo-3,5,7,8-tetrahydropyrido-
[4,3-d]pyrimidine-6-carbonyl)amino]benzoic acid (1.0 equiv),
EDC (1.5 equiv), and HOBt (1.5 equiv) were added with stirring
in dry DMF. 3-Aminomethylpyridine (1.1 equiv) was added
followed by TEA (3.0 equiv). The mixture was stirred at room
temperature overnight and purified via flash chromatography
using 0-20% DCM/MeOH. 1H NMR (DMSO-d6) δ:
9.10-9.02 (m, 2H), 8.81 (s, 1H), 8.73 (d, J=4.0 Hz, 1H), 8.29
(d, J=4.4 Hz, 1H), 7.88-7.83 (m, 1H), 7.80 (d, J=5.6 Hz, 2H),
7.60 (d, J=5.6 Hz, 2H), 4.59 (d, J=2.8 Hz, 2H), 4.31 (s, 2H),
3.70 (t, J=5.7 Hz, 2H), 2.64 (t, J=5.8 Hz, 2H), 2.31 (s, 3H).
HRMS [M þ H] calculated for C22H22N6O3 419.1834, found
419.1839.
(5) (a) Garten, A.; Petzold, S.; Korner, A.; Imai, S.-I.; Kiess, W.
Nampt: Linking NAD Biology, Metabolism and Cancer. Trends
Endocrinol. Metab. 2009, 20, 130–138. (b) Galli, M.; Van Gool, F.;
Rongvaux, A.; Andris, F.; Leo, O. The Nicotinamide Phosphoribosyl-
transferase: A Molecular Link between Metabolism, Inflammation, and
Cancer. Cancer Res. 2010, 70, 8–11. (c) Yang, T.; Sauve, A. A. NAD
Metabolism and Sirtuins: Metabolic Regulation of Protein Deacetyla-
tion in Stress and Toxicity. AAPS J. 2006, 8 (4), E632–E643.
(6) Khan, J. A.; Forouhar, F.; Tao, X.; Tong, L. Nicotinamide
Adenine Dinucleotide Metabolism as an Attractive Target for
Drug Discovery. Exp. Opin. Ther. Targets 2007, 11, 695–705.
(7) (a) FK866: Holen, K.; Saltz, L. B.; Hollywood, E.; Burk, K.;
Hanauske, A.-R. The Pharmacokinetics, Toxicities, and Biologic
Effects of FK866, a Nicotinamide Adenine Dinucleotide Biosynthe-
sis Inhibitor. Invest. New Drugs 2008, 26, 45–51. (b) GMX1778/
CHS-828:Ravaud, A.; Cerny, T.; Terret, C.; Wanders, J.; Bui, B. N.;
Hess, D.; Droz, J.-P.; Fumoleau, P.; Twelves, C. Phase I Study and
Pharmacokinetic of CHS-828, a Guanidino-Containing Compound,
Administered Orally as a Single Dose Every 3 Weeks in Solid Tumours:
An ECSG/EORTC Study. Eur. J. Cancer. 2005, 41, 702–707. Hovstadium,
P.; Larsson, R.; Jonsson, E.; Skov, T.; Kissmeyer, A.-M.; Krasilnikoff,
K.; Bergh, J.; Karlsson, M. O.; Lonnebo, A.; Ahlgren, J. A Phase I Study
of CHS 828 in Patients with Solid Tumor Malignancy. Clin. Cancer
Res. 2002, 8, 2843–2850.
(8) Colombano, G.; Travelli, C.; Galli, U.; Caldarelli, A.; Chini,
M. G.; Canonico, P., L.; Sorba, G.; Bufulco, G.; Tron, G. C.;
Genazzani, A. A. A Novel Potent Nicotinamide Phosphoribosyl-
transferase Inhibitor Synthesized via Click Chemistry. J. Med.
Chem. 2010, 53, 616–623.
(9) (a) Khan, J. A.; Tao, X.; Tong, L. Molecular Basis for the
Inhibition of Human NMPRTase, a Novel Target for Anticancer
Agents. Nat. Struct. Mol. Biol. 2006, 13, 582–588. (b) Kang, G. B.;
Bae, M.-H.; Kim, K.-M.; Im, I.; Kim, Y.-C.; Eom, S. H. Crystal
Structure of Rattus norvegicus Visfatin/PBEF/Nampt in Complex with
an FK866-Based Inhibitor. Mol. Cell 2009, 27, 667–672.
4-(2-Methyl-4-oxo-3,5,7,8-tetrahydropyrido[4,3-d]pyrimidine-
6-carbonyl)-N-(3-pyridylmethyl)benzamide (45). To a medium
vial 43 (1.0 equiv), EDC (1.5 equiv), and HOBt (1.5 equiv) were
added with stirring in dry DMF. 4-(3-Pyridylmethylcarba-
moyl)benzoic acid (1.1 equiv) was added followed by TEA
(3.0 equiv). The mixture was stirred at room temperature over-
night and purified via flash chromatography using 0-20%
DCM/MeOH. 1H NMR (DMSO-d6) δ: 9.35 (t, J = 6.5 Hz,
1H), 8.81 (s, 1H), 8.72 (d, J=2.9 Hz, 1H), 8.27 (t, J=7.0 Hz,
1H), 7.97 (d, J=4.4 Hz, 2H), 7.82 (t, J=6.9 Hz, 1H), 7.58 (d, J=
4.4 Hz, 2H), 4.63 (d, J=3.0 Hz, 2H), 4.41 (bs, 1H), 4.15 (bs, 1H),
3.91-8.83 (m, 1H), 3.56-3.44 (m, 1H), 2.69-2.59 (m, 2H), 2.28
(d, J=5.2 Hz, 3H). HRMS [M þ H] calculated for C22H21N5O3
404.1724, found 404.1720.
(10) Jones, T. R.; Thorton, T. J.; Flinn, A.; Jackman, A. L.; Newell,
D. R.; Calvert, A. H. Quinazoline Antifolates Inhibiting Thymi-
dylate Synthase: 2-Desamino Derivatives with Enhanced Solubility
and Potency. J. Med. Chem. 1989, 32, 847–852.
(11) Roswosky, A.; Forsch, R. A.; Moran, R. G. Side Chain Modified
5-Deazatolate and 5-Deazatetrahydrotolate Analogs as Mammalian
Folylpolyglutamate Synthetase and Glycinamide Ribonucleotide For-
myltransferase Inhibitors: Synthesis and in Vitro Biological Evalu-
ation. 47. N-[4-[[(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-6-yl)methyl]-
amino]benzoyl-L-glutamic Acid, a Novel A-Ring Analog of 2-Desa-
mino-5,8-dideazafolic acid. J. Med. Chem. 1992, 35, 2626–2630.
(12) Shioiri, T.; Ninomiya, K.; Yamada, S.-I. Diphenylphosphoryl
Azide. New Convenient Reagent for a Modified Curtius Reaction
and for Peptide Synthesis. J. Am. Chem. Soc. 1972, 94, 6203–6205.
(13) McLaughlin, M.; Palucki, M.; Davies, I. W. Efficient Access to
Azaindoles and Indoles. Org. Lett. 2006, 8, 3307–3310.
(14) (a) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. 5-
Arylthio Substituted 2-Amino-4-oxo-6-methylpyrrolo[2,3-d]pyri-
midine Antifolates as Thymidylate Synthase Inhibitors and Anti-
tumor Agents. J. Med. Chem. 1995, 38, 4495–4502. (b) Gangjee, A.;
Mavandadi, F.; Kisliuk, R. L.; McGuire, J. J.; Queener, S. F. 2-Amino-4-
oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as Nonclassical Antifo-
late Inhibitors of Thymidylate Synthase. J. Med. Chem. 1996, 39,
4563–4568. (c) Gangjee, A.; Jain, H. J.; Phan, J.; Lin, X.; Song, X.;
McGuire, J. J.; Kisiliuk, R. L. Dual Inhibitors of Thymidylate Synthase
and Dihydrofolate Reductase as Antitumor Agents: Design, Synthesis,
and Biological Evaluation of Classical and Nonclassical Pyrrolo[2,3-d]-
pyrimidine Antifolates. J. Med. Chem. 2006, 49, 1055–1065.
(15) Ganjee, A.; Lin, X.; Kisliuk, R. L.; McHuire, J. J. Synthesis of
N-{4-[(2,4-Diamino-5-methyl-4,7-dihydro-3H- pyrrolo[2,3-d]pyri-
midin-6-yl)thio]benzoyl}-l-glutamic Acid and N-{4-[(2-Amino-4-
oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin- 6-yl)thio]-
benzoyl}-l-glutamic Acid as Dual Inhibitors of Dihydrofolate
Reductase and Thymidylate Synthase and as Potential Antitumor
Agents. J. Med. Chem. 2005, 48, 7215–7222.
Biological Evaluation. The coupled-enzyme Nampt enzymatic
assay, PARP cellular Nampt activity assay, and HCT116 72 h
cytotoxicity assay in the presence and absence of nicotinic acid
were done as previously described.4
Acknowledgment. We thank the analytical chemistry group
of Myrexis, Inc. for the work on the purification and character-
ization of described compounds, and we thank Drs. Kraig M.
Yager and Robert Carlson for their support of this work.
Supporting Information Available: Experimental procedure
for molecular modeling. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Skelton, L. A.; Kimbell, R.; Jackman, A. L. Aminomethyl Pyridine
Analogues of the Quinazoline Antifolate ICI 198583 with a Folate-
Independent Locus of Action. In Chemistry and Biology of Pter-
idines and Folates; Pfleiderer, W., Rokos, H., Eds.; Blackwell Science:
Berlin, Germany, 1997; pp 209-212.
(2) (a) Skelton, L. A.; Ormerod, M. G.; Titley, J.; Kimbell, R.;
Brunton, L. A.; Jackman, J. L. A Novel Class of Lipophilic
Quinazoline-Based Folic Acid Analogues: Cytotoxic Agents with
a Folate-Independent Locus. Br. J. Cancer 1999, 79, 1692–1701. (b)
Skelton, L. A.; Ormerod, M. G.; Titley, J.; Jackman, J. L. Cell Cycle
Effects of CB30865, a Lipophilic Quinazoline-Based Analogue of the
Antifolate Thymidylate Synthase Inhibitor ICI 198583 with an Un-
defined Mechanism of Action. Cytometry 1998, 33, 56–66.
(16) Taylor,E.C.;Patel,H.H.;Jun,J.-G.AOne-StepRingTransformation/
Ring Annulation Approach to Pyrrolo[2,3-d]pyrimidines. A New
Synthesis of the Potent Dihydrofolate Reductase Inhibitor TNP-
351. J. Org. Chem. 1995, 60, 6684–6687.
(17) Nair, M. G.; Adapa, S. R.; Bridges, T. W. Folate Analogs. 17.
Synthesis of Pteroic Acid and 4-Amino-4-deoxypteroic Acid.
J. Org. Chem. 1981, 46, 3152–3155.
(3) Bavetsias, V.; Skelton, L. A.; Yafai, F.; Mitchell, F.; Wilson, S. C.;
Allan, B.; Jackman, A. L. The Design and Synthesis of Water-
Soluble Analogues of CB30965, a Quinazolin-4-one-Based Anti-
tumor Agent. J. Med. Chem. 2002, 45, 3692–3702.