The 27-year-old Sevilla striker is available for around £15million, but the La Liga outfit are struggling financially, and so could accept a cut-price offer for the Spaniard.
Spurs boss Andre Villas-Boas has already brought forward the summer transfer of Schalke attacking midfielder Lewis Holtby, as he bids to strengthen his White Hart Lane squad before the close of the transfer window.
Internacional's Brazilian front-man Leandro Damiao and Celtic striker Gary Hooper are both believed to have been on Tottenham's radar, but the Premier League club - who are chasing qualification for the Champions League this year - have now turned their attention back to former target Negredo.
Defoe has been playing in discomfort for some time and after a blistering start to the 2012/13 campaign, his form has suffered with only one goal in his last eight games for Spurs.
He was able to partake in training last week, but missed Sunday's FA Cup fourth round defeat to Leeds United at Elland Road.
The striker has had an injection in the hope of being fit for tomorrow's Premier League fixture at Norwich, but the fear at Tottenham is that if the problem worsens, Defoe might require an operation, leaving Villas-Boas extra short in attacking options.
With Emmanuel Adebayor away on Africa Cup of Nations duty, a new striker must now be a priority for the Portuguese tactician in the closing days before Thursday's deadline.